Compositions and methods for expressing otoferlin

ABSTRACT

Provided herein are methods and compositions for expressing Otoferlin, e.g., utilizing adeno-associated viral (AAV) particles. Such methods and compositions may be useful for treatment of diseases such as Deafness, Autosomal Recessive 9 (DFNB9).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/502,462 filed on May 5, 2017, the entire disclosure of which isincorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants EY000331,EY021721 and DC012118 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF INVENTION

Nonsyndromic deafness is a form of hearing loss that is generally causedby defects or damage to the inner ear and/or middle ear. Mutations inthe OTOF gene, which encodes the protein Otoferlin, are thought to causea type of nonsyndromic deafness called Deafness, Autosomal Recessive 9(DFNB9). Treatment of DFNB9 and other similar forms of deafnesscurrently involves using cochlear implants for severe or profoundhearing loss and hearing aids for milder forms of hearing loss. Thereremains a need for alternative treatment forms that do not rely on orrely less heavily on electronic devices for restoring hearing.

SUMMARY OF INVENTION

Provided herein are compositions and methods for expressing Otoferlin,e.g., in a cell or subject. As described herein, it has been found thatdelivery of the OTOF cDNA to otof knock-out mice via a dualadeno-associated virus (AAV) system containing different portions of theOTOF cDNA was capable of rescuing hearing in the mice to near wild-typelevels.

In some aspects, the disclosure provides a method of increasingexpression of Otoferlin in a cell, the method comprising contacting thecell with a first AAV particle comprising a first polynucleotide; andcontacting the cell with a second AAV particle comprising a secondpolynucleotide, wherein the first polynucleotide comprises invertedterminal repeat sequences flanking an expression cassette containing,from 5′ to 3′: (a) a promoter, (b) a partial coding sequence thatencodes an N-terminal portion of an Otoferlin polypeptide, (c) a splicedonor site, and (d) a first region of homology containing a sequencethat is homologous to a sequence in the second polynucleotide, and thesecond polynucleotide comprises inverted terminal repeat sequencesflanking an expression cassette containing, from 5′ to 3′: (a) a secondregion of homology containing a sequence that is homologous to asequence in the first polynucleotide, (b) a splice acceptor site, (c) apartial coding sequence that encodes a C-terminal portion of theOtoferlin polypeptide, and (d) a polyadenylation (pA) signal sequence.

In some embodiments, the region of homology in the first and secondpolynucleotides is between 50 and 500 nucleotides. In some embodiments,the region of homology in the first and second polynucleotides isbetween 50 and 300 nucleotides. In some embodiments, the region ofhomology comprises the nucleotide sequence of SEQ ID NO: 3. In someembodiments, the promoter is a chimeric CMV β actin (smcBA) promoter. Insome embodiments, the promoter comprises the sequence of SEQ ID NO: 4.In some embodiments, the Otoferlin polypeptide comprises the amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, thesplice donor site comprises the sequence of SEQ ID NO: 7. In someembodiments, the splice acceptor site comprises the sequence of SEQ IDNO: 8. In some embodiments, the inverted terminal repeat sequences areAAV2 inverted terminal repeat sequences. In some embodiments, the firstand second AAV particle are AAV2 serotype particles. In someembodiments, the cell is ex vivo. In some embodiments, the cell is invivo. In some embodiments, the cell is in a mammalian subject. In someembodiments, the subject has Deafness, Autosomal Recessive 9 (DFNB9).

In other aspects, the disclosure provides a composition comprising afirst AAV particle comprising a first polynucleotide; and a second AAVparticle comprising a second polynucleotide, wherein the firstpolynucleotide comprises inverted terminal repeat sequences flanking anexpression cassette containing, from 5′ to 3′: (a) a promoter, (b) apartial coding sequence that encodes an N-terminal portion of anOtoferlin polypeptide, (c) a splice donor site, and (d) a first regionof homology containing a sequence that is homologous to a sequence inthe second polynucleotide, and the second polynucleotide comprisesinverted terminal repeat sequences flanking an expression cassettecontaining, from 5′ to 3′: (a) a second region of homology containing asequence that is homologous to a sequence in the first polynucleotide.(b) a splice acceptor site, (c) a partial coding sequence that encodes aC-terminal portion of the Otoferlin polypeptide, and (d) apolyadenylation (pA) signal sequence.

In some embodiments, the region of homology in the first and secondpolynucleotides is between 50 and 500 nucleotides. In some embodiments,the region of homology in the first and second polynucleotides isbetween 50 and 300 nucleotides. In some embodiments, the region ofhomology comprises the nucleotide sequence of SEQ ID NO: 3. In someembodiments, the promoter is a chimeric CMV β actin (smcBA) promoter. Insome embodiments, the promoter comprises the sequence of SEQ ID NO: 4.In some embodiments, the Otoferlin polypeptide comprises the amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, thesplice donor site comprises the sequence of SEQ ID NO: 7. In someembodiments, the splice acceptor site comprises the sequence of SEQ IDNO: 8. In some embodiments, the inverted terminal repeat sequences areAAV2 inverted terminal repeat sequences. In some embodiments, the firstand second AAV particle are AAV2 serotype particles. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier.

In yet other aspects, the disclosure provides a kit comprising acomposition as described herein or comprising a first AAV particle asdescribed herein and a second AAV particle as described herein.

These and other aspects are described in more detail herein.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A is a map of a plasmid containing AAV2 inverted terminal repeats(TR) flanking a CMV enhancer, a chicken beta-actin promoter, a 5′section of the mouse Otoferlin cDNA (Otoferlin NT), a splice donorsequence (APSD), and a homologous sequence for recombination (APhead).

FIG. 1B is a map of a plasmid containing AAV2 inverted terminal repeats(TR) flanking a homologous sequence for recombination (APhead), a spliceacceptor sequence (APSA), a 3′ section of the mouse Otoferlin cDNA(Otoferlin CT), a bovine growth hormone polyadenylation signal (bGHPolyA).

FIG. 2 is a schematic of the two expression cassettes in the plasmids inFIGS. 1A and 1B.

FIG. 3A shows the annotated sequence of the expression cassette,including the inverted terminal repeats (TR) for the plasmid in FIG. 1A.

FIG. 3B shows the annotated sequence of the expression cassette,including the inverted terminal repeats (TR) for the plasmid in FIG. 1B.

FIG. 4 is a series of photographs showing expression of OTOF protein inHEK 293 cells treated with AAV2-OTOF-NT (AAV-NT) or AAV2-OTOF-NT andAAV2-OTOF-CT (AAV2-NT+CT).

FIG. 5 is a series of photographs showing expression of GFP in thecochlea organ of Corti surface preparations from wild-type mice treatedwith AAV2-GFP.

FIGS. 6A-D are a series of photographs and a graph showing OTOFexpression in the cochlea of P1-P3 mice. FIG. 6A shows expression ofOTOF protein in the mid-turn. FIG. 6B shows expression of OTOF proteinin the apex. FIG. 6C shows the difference in OTOF expression in thebase, mid-turn and apex in wild-type mice (WT, n=6) and OTOF knock-outmice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res. KO NT+CT, n=6).The left bar in each pair of bars is WT and the right bar in each pairof bars is Res. KO NT+CT. FIG. 6D shows RT-PCR data of OTOF mRNA inwild-type (WT), OTOF knock-out mice (KO) and OTOF knock-out mice treatedwith AAV2-OTOF-NT and AAV2-OTOF-CT (Res. KO).

FIGS. 7A-D show a hearing assessment in mice. FIG. 7A is a trace ofauditory brainstem response (ABR) patterns induced by auditory stimuliin wild-type mice (WT), OTOF knock-out mice either untreated (KO/KO NT)or treated (Rescued KO) with AAV2-OTOF-NT and AAV2-OTOF-CT. FIG. 7Bshows the auditory brainstem response (ABR) threshold in wild-type mice(WT), untreated Otoferlin knock-out mice (KO), Otoferlin knock-out micetreated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res KO NT+CT), and Otoferlinknock-out mice treated with AAV2-OTOF-NT (KO+NT). FIG. 7C shows a timecourse of hearing recovery in wild-type mice (WT), untreated OTOFknock-out mice (KO), Otoferlin knock-out mice treated with AAV2-OTOF-NTand AAV2-OTOF-CT (Rescued KO NT+CT), and Otoferlin knock-out micetreated with AAV2-OTOF-NT (KONT). FIG. 7D shows the click ABR thresholdin wild-type mice (WT), untreated Otoferlin knock-out mice (KO),Otoferlin knock-out mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT(Res. KO (NT+CT)), and Otoferlin knock-out mice treated withAAV2-OTOF-NT (KO+NT).

FIGS. 8A and B show Otoferlin protein expression in the OTOF rescued KOmice inner hair cells. FIG. 8A shows OTOF protein expression in P12 andolder mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT. FIG. 8B shows thepercent of inner hair cells expressing OTOF in wild-type mice (WT, n=5)and OTOF knock-out mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT(Rescued KO, n=5). The left bar in each pair of bars is WT and the rightbar in each pair of bars is Rescued KO.

FIGS. 9A and 9B are a series of graphs showing hearing assessment. FIG.9A shows ABR threshold values in wild-type mice (WT), OTOF knock-outmice (KO) and OTOF knock-out mice treated with AAV2-OTOF-NT andAAV2-OTOF-CT (Rescued KO). FIG. 9B shows hearing longevity in WT, KO andRescued KO mice.

FIG. 10 is a map of a plasmid containing AAV2 inverted terminal repeats(TR) flanking a CMV enhancer, a chicken beta-actin promoter, a 5′section of a human Otoferlin cDNA (Otoferlin NT), a splice donorsequence (APSD), and a homologous sequence for recombination (APhead).

FIG. 11 is a map of a plasmid containing AAV2 inverted terminal repeats(TR) flanking a homologous sequence for recombination (APhead), a spliceacceptor sequence (APSA), a 3′ section of a human Otoferlin cDNA(Otoferlin CT) encoding isoform 1 of Otoferlin, a bovine growth hormonepolyadenylation signal (bGH PolyA).

FIG. 12 is a map of a plasmid containing AAV2 inverted terminal repeats(TR) flanking a homologous sequence for recombination (APhead), a spliceacceptor sequence (APSA), a 3′ section of the mouse Otoferlin cDNA(Otoferlin CT) encoding isoform 5 of Otoferlin, a bovine growth hormonepolyadenylation signal (bGH PolyA).

FIG. 13 shows the annotated sequence of a human OTOF N-terminalexpression cassette, including the inverted terminal repeats (TR) forthe plasmid in FIG. 10.

FIG. 14 shows the annotated sequence of a human OTOF C-terminalexpression cassette for isoform 1, including the inverted terminalrepeats (TR) for the plasmid in FIG. 11.

FIG. 15 shows the annotated sequence of a human OTOF C-terminalexpression cassette for isoform 5, including the inverted terminalrepeats (TR) for the plasmid in FIG. 12.

DETAILED DESCRIPTION OF INVENTION

As described herein, it has been found that hearing can be restored inOtoferlin knock-out mice by treating the mice with two separate AAVparticles, one comprising the 5′ portion of the OTOF cDNA and onecomprising the 3′ portion of the OTOF cDNA and each comprising a regionof homology for promoting homologous recombination between the 5′portion and 3′ portion in vivo. This region of homology is flanked by asplice donor sequence on the 5′ side within the 5′ portion of the OTOFcDNA and a splice acceptor sequence on the 3′ side within the 3′ portionof the OTOF cDNA. Accordingly, compositions and methods are provided forincreasing expression of Otoferlin.

Exemplary Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally occurring nucleotides.

The term “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably, atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

When highly-homologous fragments are desired, the extent of percentidentity between the two sequences may be at least about 80%, preferablyat least about 85%, and more preferably about 90% or 95% or higher, asreadily determined by one or more of the sequence comparison algorithmswell-known to those of ordinary skill in the art, such as e.g., theFASTA program analysis described by Pearson and Lipman (1988).

Polynucleotides

In some aspects, polynucleotides are provided for delivering portions ofcoding sequences of an OTOF gene that encode the Otoferlin protein to acell. In some embodiments, the coding sequences are derived from a humanOTOF gene (see, e.g., NCBI Gene ID: 9381 and cDNA sequencesNM_001287489.1, NM_004802.3, NM_194248.2, NM_194322.2, and NM_194323.2).In some embodiments, the coding sequences are derived from a mouse OTOFgene (see, e.g., NCBI Gene ID 83762 and cDNA sequences NM_001100395.1,NM_001286421.1, NM_001313767.1, and NM_031875.2). In some embodiments, afirst and a second polynucleotide are provided. It is to be understoodthat “first,” “second,” “third,” and the like are not meant to imply aparticular order or importance unless expressly stated otherwise.

In some embodiments, the first polynucleotide comprises invertedterminal repeat sequences flanking an expression cassette containing,from 5′ to 3′, one or more of (a) a promoter, (b) a partial codingsequence that encodes an N-terminal portion of an Otoferlin polypeptide,(c) a splice donor site, and (d) a first region of homology containing asequence that is homologous to a sequence in the second polynucleotide.In some embodiments, the first polynucleotide comprises at least two, atleast three or all four of (a), (b), (c), and (d).

In some embodiments, the second polynucleotide comprises invertedterminal repeat sequences flanking an expression cassette containing,from 5′ to 3′, one or more of (a) a second region of homology containinga sequence that is homologous to a sequence in the first polynucleotide,(b) a splice acceptor site, (c) a partial coding sequence that encodes aC-terminal portion of the Otoferlin polypeptide, and (d) apolyadenylation (pA) signal sequence. In some embodiments, the secondpolynucleotide comprises at least two, at least three or all four of(a), (b), (c), and (d).

The partial coding sequences contained within the polynucleotidesdescribed herein may be designed so that, upon delivery of thepolynucleotides, the partial coding sequences are joined together, e.g.,through homologous recombination, and form a complete coding sequencethat encodes an Otoferlin polypeptide.

In some embodiments, the polynucleotides are plasmids (e.g., a circularnucleic acid comprising one or more of an origin of replication, aselectable marker, and a reporter gene). In some embodiments,polynucleotides described herein, such as a plasmid, may also containmarker or reporter genes, e.g., LacZ or a fluorescent protein, and anorigin of replication. In some embodiments, the plasmid is transfectedinto a producer cell that produces AAV particles containing theexpression cassettes contained within the plasmids.

In some embodiments, the polynucleotides are nucleic acid vectors suchas a recombinant adeno-associated virus (AAV) vectors. Exemplary AAVnucleic acid vectors useful according to the disclosure includesingle-stranded (ss) or self-complementary (sc) AAV nucleic acidvectors.

In some embodiments, recombinant AAV particles comprise thepolynucleotides, such as a single-stranded (ss) or self-complementary(sc) AAV nucleic acid vectors. In some embodiments, the polynucleotidescontain expression constructs as described herein and inverted terminalrepeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITRsequences) flanking the expression constructs. In some embodiments, thepolynucleotides are encapsidated by viral capsids.

Accordingly, in some embodiments, an AAV particle comprises a viralcapsid and a polynucleotide as described herein, which is encapsidatedby the viral capsid. In some embodiments, the viral capsid comprises 60capsid protein subunits comprising VP1, VP2 and VP3. In someembodiments, the VP1, VP2, and VP3 subunits are present in the capsid ata ratio of approximately 1:1:10, respectively.

In some embodiments, polynucleotides as described herein (e.g., firstand second polynucleotides) comprise regions of homology, e.g., topromote homologous recombination between the polynucleotides oncedelivered to a cell (see, e.g., Ghosh et al. Efficient transgenereconstitution with hybrid dual AAV vectors carrying the minimizedbridging sequences. Hum Gene Ther. 2011 January; 22(1):77-83). In someembodiments, a first region of homology and a second region of homologyhave a threshold level of sequence identity with each other in order topromote homologous recombination. In some embodiments the first regionof homology has at least 75%, at least 80%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identitywith the second region of homology. Unless otherwise specified, as usedherein percent sequence identity and/or similarity of two sequences canbe determined using the algorithm of Karlin and Altschul (1990),modified as in Karlin and Altschul (1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990). BLAST searches can be performed with the NBLAST program,score=100, word-length=12, to obtain sequences with the desired percentsequence identity. To obtain gapped alignments for comparison purposes,Gapped BLAST can be used as described (Altschul et al., 1997). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (NBLAST and XBLAST) can be used in accordance withpublished methods. In some embodiments, each region of homology isindependently between 50 and 500, 50 and 400, 50 and 300, 100 and 500,100 and 400, 100 and 300, 200 and 500, 200 and 400, or 200 and 300nucleotides. In some embodiments, the regions of homology are identicaland each region of homology is between 50 and 500, 50 and 400, 50 and300, 100 and 500, 100 and 400, 100 and 300, 200 and 500, 200 and 400, or200 and 300 nucleotides. In some embodiments, the region homologycomprises a sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical with the nucleotide sequence

(SEQ ID NO: 3) CCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTC.

In some embodiments, polynucleotides described herein may comprise oneor more regulatory elements. A person of ordinary skill in the art canselect regulatory elements for use in appropriate host cells, forexample, mammalian or human host cells. Regulatory elements include, forexample, promoters, transcription termination sequences, translationtermination sequences, enhancers, and polyadenylation elements. Apolynucleotide described herein may comprise a promoter sequenceoperably linked to a nucleotide sequence encoding a desired polypeptide,such as Otoferlin. Promoters contemplated for use in the subjectinvention include, but are not limited to, cytomegalovirus (CMV)promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, chimericCMV/chicken β actin promoter (CBA) and the truncated form of CBA (smCBA)(see, e.g., Haire et al. 2006 and U.S. Pat. No. 8,298,818, which isspecifically incorporated herein in its entirety by express referencethereto). In some embodiments, the promoter is the truncated chimericCMV β actin (smcBA) promoter. In some embodiments, the promotercomprises a sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical with the nucleotide sequence

(SEQ ID NO: 4) GGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAG CGCGCGGCGGGCG.

In some embodiments, polynucleotides as described herein comprise apartial coding sequence that encodes an N-terminal or C-terminal portionof an Otoferlin polypeptide, wherein the partial coding sequences can bespliced or otherwise combined together in vivo in order to encode anOtoferlin polypeptide. In some embodiments, the Otoferlin polypeptide isa human Otoferlin polypeptide. In some embodiments, the Otoferlinpolypeptide is a long isoform of a human Otoferlin polypeptide (see,e.g., Yasunaga et al. OTOF Encodes Multiple Long and Short Isoforms:Genetic Evidence That the Long Ones Underlie Recessive Deafness DFNB9.Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, the Otoferlinpolypeptide comprises a sequence that is at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical with one or both of thefollowing amino acid sequences:

Human OTOF isoform 1-Genbank Number AF183185.1 (SEQ ID NO: 5)MALLIHLKTVSELRGRGDRIAKVTFRGQSFYSRVLENCEDVADFDETFRWPVASSIDRNEMLEIQVFNYSKVFSNKLIGTFRMVLQKVVEESHVEVTDTLIDDNNAIIKTSLCVEVRYQATDGTVGSWDDGDFLGDESLQEEEKDSQETDGLLPGSRPSSRPPGEKSFRRAGRSVFSAMKLGKNRSHKEEPQRPDEPAVLEMEDLDHLAIRLGDGLDPDSVSLASVTALTTNVSNKRSKPDIKMEPSAGRPMDYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEYFVFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQFHHKWAILSDPDDISSGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEGNLLLPEGVPPERQWARFYVKIYRAEGLPRMNTSLMANVKKAFIGENKDLVDPYVQVFFAGQKGKTSVQKSSYEPLWNEQVVFTDLFPPLCKRMKVQIRDSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMYGSTRNYTLLDEHQDLNEGLGEGVSFRARLLLGLAVEIVDTSNPELTSSTEVQVEQATPISESCAGKMEEFFLFGAFLEASMIDRRNGDKPITFEVTIGNYGNEVDGLSRPQRPRPRKEPGDEEEVDLIQNASDDEAGDAGDLASVSSTPPMRPQVTDRNYFHLPYLERKPCIYIKSWWPDQRRRLYNANIMDHIADKLEEGLNDIQEMIKTEKSYPERRLRGVLEELSCGCCRFLSLADKDQGHSSRTRLDRERLKSCMRELENMGQQARMLRAQVKRHTVRDKLRLCQNFLQKLRFLADEPQHSIPDIFIWMMSNNKRVAYARVPSKDLLFSIVEEETGKDCAKVKTLFLKLPGKRGFGSAGWTVQAKVELYLWLGLSKQRKEFLCGLPCGFQEVKAAQGLGLHAFPPVSLVYTKKQAFQLRAHMYQARSLFAADSSGLSDPFARVFFINQSQCTEVLNETLCPTWDQMLVFDNLELYGEAHELRDDPPIIVIEIYDQDSMGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYRGNATAGDLLAAFELLQIGPAGKADLPPINGPVDVDRGPIMPVPMGIRPVLSKYRVEVLFWGLRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEVDLPENELLHPPLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPSWNTTVRLLRRCRVLCNGGSSSHSTGEVVVTMEPEVPIKKLETMVKLDATSEAVVKVDVAEEEKEKKKKKKGTAEEPEEEEPDESMLDWWSKYFASIDTMKEQLRQQEPSGIDLEEKEEVDNTEGLKGSMKGKEKARAAKEEKKKKTQSSGSGQGSEAPEKKKPKIDELKVYPKELESEFDNFEDWLHTFNLLRGKTGDDEDGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDSTYGMFQGIPSNDPINVLVRVYVVRATDLHPADINGKADPYIAIRLGKTDIRDKENYISKQLNPVFGKSFDIEASFPMESMLTVAVYDWDLVGTDDLIGETKIDLENRFYSKHRATCGIAQTYSTHGYNIWRDPMKPSQILTRLCKDGKVDGPHFGPPGRVKVANRVFTGPSEIEDENGQRKPTDEHVALLALRHWEDIPRAGCRLVPEHVETRPLLNPDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIIWNTDEVVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWRYLFPFDYLAAEEKIVISKKESMFSWDETEYKIPARLTLQIWDADHFSADDFLGAIELDLNRFPRGAKTAKQCTMEMATGEVDVPLVSIFKQKRVKGWWPLLARNENDEFELTGKVEAELHLLTAEEAEKNPVGLARNEPDPLEKPNRPDTSFIWFLNPLKSARYFLWHTYRWLLLKLLLLLLLLLLLALFLYSVPGYLVKKILGAHuman OTOF isoform 5-Genbank Number NP_001274418 (SEQ ID NO: 6)MALLIHLKTVSELRGRGDRIAKVTFRGQSFYSRVLENCEDVADFDETFRWPVASSIDRNEMLEIQVFNYSKVFSNKLIGTFRMVLQKVVEESHVEVTDTLIDDNNAIIKTSLCVEVRYQATDGTVGSWDDGDFLGDESLQEEEKDSQETDGLLPGSRPSSRPPGEKSFRRAGRSVFSAMKLGKNRSHKEEPQRPDEPAVLEMEDLDHLAIRLGDGLDPDSVSLASVTALTTNVSNKRSKPDIKMEPSAGRPMDYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEYFVFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQFHHKWAILSDPDDISSGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEGNLLLPEGVPPERQWARFYVKIYRAEGLPRMNTSLMANVKKAFIGENKDLVDPYVQVFFAGQKGKTSVQKSSYEPLWNEQVVFTDLFPPLCKRMKVQIRDSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMYGSTRNYTLLDEHQDLNEGLGEGVSFRARLLLGLAVEIVDTSNPELTSSTEVQVEQATPISESCAGKMEEFFLFGAFLEASMIDRRNGDKPITFEVTIGNYGNEVDGLSRPQRPRPRKEPGDEEEVDLIQNASDDEAGDAGDLASVSSTPPMRPQVTDRNYFHLPYLERKPCIYIKSWWPDQRRRLYNANIMDHIADKLEEGLNDIQEMIKTEKSYPERRLRGVLEELSCGCCRFLSLADKDQGHSSRTRLDRERLKSCMRELENMGQQARMLRAQVKRHTVRDKLRLCQNFLQKLRFLADEPQHSIPDIFIWMMSNNKRVAYARVPSKDLLFSIVEEETGKDCAKVKTLFLKLPGKRGFGSAGWTVQAKVELYLWLGLSKQRKEFLCGLPCGFQEVKAAQGLGLHAFPPVSLVYTKKQAFQLRAHMYQARSLFAADSSGLSDPFARVFFINQSQCTEVLNETLCPTWDQMLVFDNLELYGEAHELRDDPPIIVIEIYDQDSMGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYRGNATAGDLLAAFELLQIGPAGKADLPPINGPVDVDRGPIMPVPMGIRPVLSKYRVEVLFWGLRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEVDLPENELLHPPLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPSWNTTVRLLRRCRVLCNGGSSSHSTGEVVVTMEPEVPIKKLETMVKLDATSEAVVKVDVAEEEKEKKKKKKGTAEEPEEEEPDESMLDWWSKYFASIDTMKEQLRQQEPSGIDLEEKEEVDNTEGLKGSMKGKEKARAAKEEKKKKTQSSGSGQGSEAPEKKKPKIDELKVYPKELESEFDNFEDWLHTFNLLRGKTGDDEDGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDSTYGMFQGIPSNDPINVLVRVYVVRATDLHPADINGKADPYIAIRLGKTDIRDKENYISKQLNPVFGKSFDIEASFPMESMLTVAVYDWDLVGTDDLIGETKIDLENRFYSKHRATCGIAQTYSTHGYNIWRDPMKPSQILTRLCKDGKVDGPHFGPPGRVKVANRVFTGPSEIEDENGQRKPTDEHVALLALRHWEDIPRAGCRLVPEHVETRPLLNPDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIIWNTDEVVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWRYLFPFDYLAAEEKIVISKKESMFSWDETEYKIPARLTLQIWDADHFSADDFLGAIELDLNRFPRGAKTAKQCTMEMATGEVDVPLVSIFKQKRVKGWWPLLARNENDEFELTGKVEAELHLLTAEEAEKNPVGLARNEPDPLEKPNRPDTAFVWFLNPLKSIKYLICTRYKWLIIKIVLALLGLLMLGLFLYSLPGYMVKKLLGA

In some embodiments, the Otoferlin polypeptide is a mouse Otoferlinpolypeptide. In some embodiments, the Otoferlin polypeptide comprises asequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical with the following amino acid sequence:

Mouse OTOF Isoform 1-Genbank Number NP_001093865.1 (SEQ ID NO: 9)MALIVHLKTVSELRGKGDRIAKVTFRGQSFYSRVLENCEGVADFDETFRWPVASSIDRNEVLEIQIFNYSKVFSNKLIGTFCMVLQKVVEENRVEVTDTLMDDSNAIIKTSLSMEVRYQATDGTVGPWDDGDFLGDESLQEEKDSQETDGLLPGSRPSTRISGEKSFRSKGREKTKGGRDGEHKAGRSVFSAMKLGKTRSHKEEPQRQDEPAVLEMEDLDHLAIQLGDGLDPDSVSLASVTALTSNVSNKRSKPDIKMEPSAGRPMDYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEYFVFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQFHHKWAILSDPDDISAGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEGNLLLPEGVPPERQWARFYVKIYRAEGLPRMNTSLMANVKKAFIGENKDLVDPYVQVFFAGQKGKTSVQKSSYEPLWNEQVVFTDLFPPLCKRMKVQIRDSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMYGSTRNYTLLDEHQDLNEGLGEGVSFRARLMLGLAVEILDTSNPELTSSTEVQVEQATPVSESCTGRMEEFFLFGAFLEASMIDRKNGDKPITFEVTIGNYGNEVDGMSRPLRPRPRKEPGDEEEVDLIQNSSDDEGDEAGDLASVSSTPPMRPQITDRNYFHLPYLERKPCIYIKSWWPDQRRRLYNANIMDHIADKLEEGLNDVQEMIKTEKSYPERRLRGVLEELSCGCHRFLSLSDKDQGRSSRTRLDRERLKSCMRELESMGQQAKSLRAQVKRHTVRDKLRSCQNFLQKLRFLADEPQHSIPDVFIWMMSNNKRIAYARVPSKDLLFSIVEEELGKDCAKVKTLFLKLPGKRGFGSAGWTVQAKLELYLWLGLSKQRKDFLCGLPCGFEEVKAAQGLGLHSFPPISLVYTKKQAFQLRAHMYQARSLFAADSSGLSDPFARVFFINQSQCTEVLNETLCPTWDQMLVFDNLELYGEAHELRDDPPIIVIEIYDQDSMGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYRGSATAGDLLAAFELLQIGPSGKADLPPINGPVDMDRGPIMPVPVGIRPVLSKYRVEVLFWGLRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEVDLPENELLHPPLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPNWNTTGEVVVSMEPEEPVKKLETMVKLDATSDAVVKVDVAEDEKERKKKKKKGPSEEPEEEEPDESMLDWWSKYFASIDTMKEQLRQHETSGTDLEEKEEMESAEGLKGPMKSKEKSRAAKEEKKKKNQSPGPGQGSEAPEKKKAKIDELKVYPKELESEFDSFEDWLHTFNLLRGKTGDDEDGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDPTYGMFQGIPSNDPINVLVRIYVVRATDLHPADINGKADPYIAIKLGKTDIRDKENYISKQLNPVFGKSFDIEASFPMESMLTVAVYDWDLVGTDDLIGETKIDLENRFYSKHRATCGIAQTYSIHGYNIWRDPMKPSQILTRLCKEGKVDGPHFGPHGRVRVANRVFTGPSEIEDENGQRKPTDEHVALSALRHWEDIPRVGCRLVPEHVETRPLLNPDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIVWNTDEVVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWRYLFPFDYLAAEEKIVMSKKESMFSWDETEYKIPARLTLQIWDADHFSADDFLGAIELDLNRFPRGAKTAKQCTMEMATGEVDVPLVSIFKQKRVKGWWPLLARNENDEFELTGKVEAELHLLTAEEAEKNPVGLARNEPDPLEKPNRPDTAFVWFLNPLKSIKYLICTRYKWLIIKIVLALLGLLMLALFLYSLPGYMVKKLLGA

In some embodiments, polynucleotides described herein comprise a splicedonor or splice acceptor site. In some embodiments, the splice donorand/or splice acceptor sites contain splice consensus sequences. In someembodiments, the splice donor and/or splice acceptor sites containsequences splice consensus sequences derived from alkaline phosphatase.In some embodiments, the splice donor site comprises a sequence that isat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identicalwith the nucleotide sequenceGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGA (SEQ ID NO: 7). In some embodiments, thesplice acceptor site comprises a sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical with the nucleotidesequence

(SEQ ID NO: 8) TAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG.

In some embodiments, polynucleotides described herein comprise ITRsequences. The ITR sequences of a polynucleotide described herein can bederived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)or can be derived from more than one serotype. In some embodiments ofthe polynucleotide provided herein, the ITR sequences are derived fromAAV2. ITR sequences and plasmids containing ITR sequences are known inthe art and commercially available (see, e.g., products and servicesavailable from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, SanDiego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene,Cambridge, Mass.; and Gene delivery to skeletal muscle results insustained expression and systemic delivery of a therapeutic protein.Kessler P D, Podsakoff G M, Chen X, McQuiston S A, Colosi P C, Matelis LA, Kurtzman G J, Byrne B J. Proc Natl Acad Sci USA. 1996 Nov. 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™.Viral Vectors for Gene Therapy Methods and Protocols.10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. TargetedIntegration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M.Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos.5,139,941 and 5,962,313, all of which are incorporated herein byreference). An exemplary AAV2 ITR sequence for flanking the 5′ end of anexpression construct comprises the sequence:TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC (SEQ ID NO: 10). An exemplary AAV2 ITRsequence for flanking the 3′ end of an expression construct comprisesthe sequence

(SEQ ID NO: 11) ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACC.

In some embodiments, polynucleotides described herein may furtheroptionally include one or more transcription termination sequences, oneor more translation termination sequences, one or more signal peptidesequences, one or more internal ribosome entry sites (IRES), and/or oneor more enhancer elements, or any combination thereof. Transcriptiontermination regions can typically be obtained from the 3′ untranslatedregion of a eukaryotic or viral gene sequence. Transcription terminationsequences can be positioned downstream of a coding sequence to providefor efficient termination. Signal peptide sequences are amino-terminalpeptidic sequences that encode information responsible for the locationof an operably-linked polypeptide to one or more post-translationalcellular destinations, including, for example, specific organellecompartments, or to the sites of protein synthesis and/or activity, andeven to the extracellular environment. In some embodiments, apolynucleotide as described herein comprises a bovine growth hormonepolyadenylation signal.

In some embodiments, the expression constructs contained within thepolynucleotides described herein are no more than 5 kilobases, no morethan 4 kilobases, or no more than 3 kilobases in size. In someembodiments, the expression construct is between 4 and 5 kilobases insize.

In some embodiments, polynucleotides described herein are containedwithin one or more recombinant AAV particles (e.g., first and second AAVparticles). The AAV particles may be of any AAV serotype (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10), including any derivative (includingnon-naturally occurring variants of a serotype) or pseudotype.Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17,AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41,AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83,AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, andAAVr3.45. Such AAV serotypes and derivatives/pseudotypes, and methods ofproducing such derivatives/pseudotypes are known in the art (see, e.g.,Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012Jan. 24. The AAV vector toolkit: poised at the clinical crossroads.Asokan Al, Schaffer D V, Samulski R J.). In some embodiments, the firstand second AAV particle are AAV2 serotype particles.

Methods of producing AAV particles and polynucleotides are known in theart and commercially available (see, e.g., Zolotukhin et al. Productionand purification of serotype 1, 2, and 5 recombinant adeno-associatedviral vectors. Methods 28 (2002) 158-167; and U.S. Patent PublicationNumbers US20070015238 and US20120322861, which are incorporated hereinby reference; and plasmids and kits available from ATCC and CellBiolabs, Inc.). For example, the polynucleotides (e.g., as plasmids) maybe combined with one or more helper plasmids, e.g., that contain a repgene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene(encoding VP1, VP2, and VP3), and transfected into a producer cell linesuch that the AAV particle can be packaged and subsequently purified.

In some embodiments, the one or more helper plasmids includes a firsthelper plasmid comprising a rep gene and a cap gene and a second helperplasmid comprising other genes that assist in AAV production, such as aE1a gene, a E1b gene, a E4 gene, a E2a gene, and a VA gene. In someembodiments, the rep gene is a rep gene derived from AAV2. Helperplasmids, and methods of making such plasmids, are known in the art andcommercially available (see, e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs,pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids fromPlasmidFactory, Bielefeld, Germany; other products and servicesavailable from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, SanDiego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene,Cambridge, Mass.; pxx6; Grimm et al. (1998), Novel Tools for Productionand Purification of Recombinant Adenoassociated Virus Vectors, HumanGene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identificationof a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids,Journal of Virology, Vol. 77, 11072-11081; Grimm et al. (2003), HelperVirus-Free, Optically Controllable, and Two-Plasmid-Based Production ofAdeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy,Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change inthe Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of HiddenVP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P.and Snyder, R. O. (2008), International efforts for recombinantadenoassociated viral vector reference standards, Molecular Therapy,Vol. 16, 1185-1188).

An exemplary, non-limiting, AAV particle production method is describednext. One or more helper plasmids are produced or obtained, whichcomprise rep and cap ORFs for the desired AAV serotype and theadenoviral VA, E2A (DBP), and E4 genes under the transcriptional controlof their native promoters. HEK293 cells (available from ATCC®) aretransfected via CaPO₄-mediated transfection, lipids or polymericmolecules such as Polyethylenimine (PEI) with the helper plasmid(s) anda plasmid containing a polynucleotide described herein. Alternatively,in another non-limiting example, Sf9-based producer stable cell linesare infected with a single recombinant baculovirus containing thepolynucleotide. As a further non-limiting alternative, in anotherexample HEK293 or BHK cell lines are infected with a HSV containing thepolynucleotide and optionally one or more helper HSVs containing rep andcap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4genes under the transcriptional control of their native promoters. TheHEK293, BHK, or Sf9 cells are then incubated for at least 60 hours toallow for AAV particle production. The AAV particles can then bepurified using any method known in the art or described herein, e.g., byiodixanol step gradient, CsCl gradient, chromatography, or polyethyleneglycol (PEG) precipitation.

The disclosure also contemplates host cells that comprise at least oneof the disclosed AAV particles or polynucleotides. Such host cellsinclude mammalian host cells, with human host cells being preferred, andmay be either isolated, in cell or tissue culture. In the case ofgenetically modified animal models (e.g., a mouse), the transformed hostcells may be comprised within the body of a non-human animal itself.

Methods and Subjects

In some aspects, methods of increasing expression of Otoferlin in a cellare provided. In some embodiments, the method comprises contacting thecell with a first AAV particle as described herein comprising a firstpolynucleotide as described herein; and contacting the cell with asecond AAV particle as described herein comprising a secondpolynucleotide as described herein. In some embodiments, the cell is amammalian cell such as a mouse or human cell. In some embodiments, thecell is ex vivo. In some embodiments, the cell is in vivo. In someembodiments, the cell is a cell of the ear (e.g., the cell of a humanear). In some embodiments, the cell is a cell of the inner ear (e.g.,the cell of a human inner ear). In some embodiments, the cell is in asubject (e.g., a mammalian subject such as a human subject).

Other aspects of the disclosure relate to treatment of a disease orcondition caused by decreased or absent expression or activity ofOtoferlin. In some embodiments, the method comprises administering to asubject a therapeutically effective amount of a first AAV particle asdescribed herein comprising a first polynucleotide as described hereinand a therapeutically effective amount of a second AAV particle asdescribed herein comprising a second polynucleotide as described herein.In some embodiments, the subject is a human subject and the subject hasDeafness, Autosomal Recessive 9 (DFNB9). In some embodiments, thesubject is a human subject having impaired vestibular function or avestibular disorder (see, e.g., Dulon et al. Otoferlin is Critical for aHighly Sensitive and Linear Calcium Dependent Exocytosis at VestibularHair Cell Ribbon Synapses. J Neurosci. 2009; 29(34): 10474-10487).

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject. The compositions described above orelsewhere herein are typically administered to a subject in an effectiveamount, that is, an amount capable of producing a desirable result. Thedesirable result will depend upon the active agent being administered.For example, an effective amount of AAV particles may be an amount ofthe particles that are capable of transferring an expression constructto a host organ, tissue, or cell. A therapeutically acceptable amountmay be an amount that is capable of treating a disease, e.g., DFNB9. Asis well known in the medical and veterinary arts, dosage for any onesubject depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, theactive ingredient(s) in the composition, time and route ofadministration, general health, and other drugs being administeredconcurrently.

The AAV particles or polynucleotides may be delivered in the form of acomposition, such as a composition comprising the active ingredient,such as AAV particles described herein, and a pharmaceuticallyacceptable carrier as described herein. The AAV particles orpolynucleotides may be prepared in a variety of compositions, and mayalso be formulated in appropriate pharmaceutical vehicles foradministration to human or animal subjects. In some embodiments, wherefirst and second AAV particles are utilized, the first and second AAVparticles may be contained within the same composition or withindifferent compositions and may be administered together or separately.

In some embodiments, the AAV particles administered to a subject may beprovided in a composition having a concentration on the order rangingfrom 10⁶ to 10¹⁴ particles/ml or 10³ to 10¹⁵ particles/ml, or any valuesthere between for either range, such as for example, about 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ particles/ml. In oneembodiment, AAV particles of higher than 10¹³ particles/ml are beadministered. In some embodiments, the number of AAV particlesadministered to a subject may be on the order ranging from 10⁶ to 10¹⁴vector genomes(vgs)/ml or 10³ to 10¹⁵ vgs/ml, or any values therebetweenfor either range, such as for example, about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, or 10¹⁴ vgs/ml. In one embodiment, AAV particles ofhigher than 10¹³ vgs/ml are be administered. The AAV particles can beadministered as a single dose, or divided into two or moreadministrations as may be required to achieve therapy of the particulardisease or disorder being treated. In some embodiments, 0.0001 ml to 10mls are delivered to a subject. In some embodiments, the number of AAVparticles administered to a subject may be on the order ranging from10⁶-10¹⁴ vg/kg, or any values therebetween, such as for example, about10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ vgs/kg. In someembodiments, when a first AAV particle comprising a first polynucleotideas described herein and second AAV particle comprising a secondpolynucleotide as described herein are administered, the amountadministered is the same for both particles. In some embodiments, when afirst AAV particle comprising a first polynucleotide as described hereinand second AAV particle comprising a second polynucleotide as describedherein are administered, the amount administered is different for eachparticle.

If desired, AAV particles may be administered in combination with otheragents or treatments as well, such as, e.g., proteins or polypeptides orvarious pharmaceutically-active agents, including one or more systemicor topical administrations of therapeutic polypeptides, biologicallyactive fragments, or variants thereof. In fact, there is virtually nolimit to other components that may also be included, given that theadditional agents do not cause a significant adverse effect upon contactwith the target cells or host tissues. The AAV particles may thus bedelivered along with various other agents or treatments as required inthe particular instance. In some embodiments, AAV particle treatment maybe accompanied by use of a hearing aid.

In certain circumstances it will be desirable to deliver the AAVparticles in suitably formulated pharmaceutical compositions disclosedherein either subcutaneously, parenterally, intravenously,intramuscularly, intraperitoneally, by oral or nasal inhalation, or bydirect injection to one or more cells, tissues, or organs. In someembodiments, the administration is a route suitable for systemicdelivery, such as by intravenous injection or infusion. In someembodiments, the administration is to the ear, e.g., via intra-cochlearadministration. The pharmaceutical forms of the AAV particlecompositions suitable for injectable use include sterile aqueoussolutions or dispersions. In some embodiments, the form is sterile andfluid to the extent that easy syringability exists. In some embodiments,the form is stable under the conditions of manufacture and storage andis preserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, saline, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, intravitreal, subretinal, subcutaneous andintraperitoneal administration. In this connection, a sterile aqueousmedium that can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,and the general safety and purity standards as required by, e.g., FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the AAVparticles in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization or another sterilization technique. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

The amount of AAV particle or polynucleotide compositions and time ofadministration of such compositions will be within the purview of theskilled artisan having benefit of the present teachings. It is likely,however, that the administration of therapeutically effective amounts ofthe disclosed compositions may be achieved by a single administration,such as for example, a single injection of sufficient numbers ofinfectious particles to provide therapeutic benefit to the patientundergoing such treatment. Alternatively, in some circumstances, it maybe desirable to provide multiple, or successive administrations of theAAV particle compositions, either over a relatively short, or arelatively prolonged period of time, as may be determined by the medicalpractitioner overseeing the administration of such compositions.

The composition may include AAV particles, either alone, or incombination with one or more additional active ingredients, which may beobtained from natural or recombinant sources or chemically synthesized.

Toxicity and efficacy of the compositions utilized in methods of thedisclosure can be determined by standard pharmaceutical procedures,using either cells in culture or experimental animals to determine theLD50 (the dose lethal to 50% of the population). The dose ratio betweentoxicity and efficacy is the therapeutic index and it can be expressedas the ratio LD50/ED50. Those compositions that exhibit largetherapeutic indices are preferred. While those that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat minimizes the potential damage of such side effects. The dosage ofcompositions as described herein lies generally within a range thatincludes an ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized.

Aspects of the disclosure relate to methods for use with a subject, suchas human or non-human primate subjects. Non-limiting examples ofnon-human primate subjects include macaques (e.g., cynomolgus or rhesusmacaques), marmosets, tamarins, spider monkeys, owl monkeys, vervetmonkeys, squirrel monkeys, baboons, gorillas, chimpanzees, andorangutans. In some embodiments, the subject is a human subject. Otherexemplary subjects include domesticated animals such as dogs and cats;livestock such as horses, cattle, pigs, sheep, goats, and chickens; andother animals such as mice, rats, guinea pigs, and hamsters.

In some embodiments, the subject has or is suspected of having a diseasethat may be treated with gene therapy. In some embodiments, the subjecthas or is suspected of having Deafness, Autosomal Recessive 9 (DFNB9).DFNB9 is an autosomal recessive form of deafness though to be caused bymutations in the OTOF gene that result in a decrease in expression,functionality, or both, of the Otoferlin protein. Otoferlin protein hasbeen shown to be important for exocytosis at the auditory ribbon synapse(see, e.g., Roux et al. Otoferlin, defective in a human deafness form,is essential for exocytosis at the auditory ribbon synapse. (2006) Cell127(2):277-89). Subjects having DFNB9 can be identified by the skilledphysician, e.g., using a combination of electrophysiologic testing ofauditory brain stem responses (ABRs) and genetic testing to identifymutations in the OTOF gene (see, e.g., OMIM entries 603681 and 601071).In some embodiments, the subject is a human subject that has one or moreof the following nonsense or missense mutations in the OTOF gene:TYR730TER, GLN829TER, PRO1825ALA, PRO50ARG, LEU1011PRO, ILE515THR,ARG1939GLN, or GLY541SER. In some embodiments, the subject is a humansubject that has an A-to-G transition at the intron 8/exon 9 junction(IVS8-2A-G) or an G-to-A transition at position+1, the first intronicnucleotide in the splice donor site of exon 5 or a G-C transversion inthe donor splice site of intron 39. In some embodiments, the subject isa human subject that has a one base pair deletion (1778G) in exon 16,leading to a stop codon, and a 6141G-A change, resulting in anARG-to-GLN substitution in exon 48.

Compositions

Other aspects of the disclosure relate to compositions comprising AAVparticles or polynucleotides described herein. In some embodiments, AAVparticles described herein are added to a composition, e.g., apharmaceutical composition.

In some embodiments, the composition comprises a pharmaceuticallyacceptable carrier. The term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the AAV particles are administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum oil such as mineral oil, vegetableoil such as peanut oil, soybean oil, and sesame oil, animal oil, or oilof synthetic origin. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers. Non-limiting examplesof pharmaceutically acceptable carriers include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup,methylcellulose, ethylcellulose, hydroxypropylmethylcellulose,polyacrylic acids, lubricating agents (such as talc, magnesium stearate,and mineral oil), wetting agents, emulsifying agents, suspending agents,preserving agents (such as methyl-, ethyl-, andpropyl-hydroxy-benzoates), and pH adjusting agents (such as inorganicand organic acids and bases). Other examples of carriers includephosphate buffered saline, HEPES-buffered saline, and water forinjection, any of which may be optionally combined with one or more ofcalcium chloride dihydrate, disodium phosphate anhydrous, magnesiumchloride hexahydrate, potassium chloride, potassium dihydrogenphosphate, sodium chloride, or sucrose. Other examples of carriers thatmight be used include saline (e.g., sterilized, pyrogen-free saline),saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer,and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (for example, serum albumin), EDTA, sodiumchloride, liposomes, mannitol, sorbitol, and glycerol. USP gradecarriers and excipients are particularly useful for delivery of AAVparticles to human subjects. Such compositions may further optionallycomprise a liposome, a lipid, a lipid complex, a microsphere, amicroparticle, a nanosphere, or a nanoparticle, or may be otherwiseformulated for administration to the cells, tissues, organs, or body ofa subject in need thereof. Methods for making such compositions are wellknown and can be found in, for example, Remington: The Science andPractice of Pharmacy, 22^(nd) edition, Pharmaceutical Press, 2012.

Typically, such compositions may contain at least about 0.1% of thetherapeutic agent (e.g., AAV particles) or more, although the percentageof the active ingredient(s) may, of course, be varied and mayconveniently be between about 1 or 2% and about 70% or 80% or more ofthe weight or volume of the total formulation. Naturally, the amount oftherapeutic agent(s) (e.g., AAV particles) in eachtherapeutically-useful composition may be prepared in such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In some embodiments, a composition described herein may be administeredto a subject in need thereof, such as a subject having DFNB9. In someembodiments, a method described herein may comprise administering acomposition or multiple compositions comprising AAV particles asdescribed herein to a subject in need thereof. In some embodiments, thesubject is a human subject. In some embodiments, the subject has or issuspected of having a disease that may be treated with gene therapy,such as DFNB9. In some embodiments, the subject has been diagnosed withDFNB9.

Kits

Other aspects of the disclosure relate to kits comprising AAV particlesor polynucleotides as described herein in one or more containers. Kitscan optionally include pharmaceutically acceptable carriers and/ordiluents. In some embodiments, the kit includes instructions orpackaging materials that describe how to administer AAV particles orpolynucleotides contained within the kit to a selected cell orrecipient. Containers of the kit can be of any suitable material, e.g.,glass, plastic, metal, etc., and of any suitable size, shape, orconfiguration. In some embodiments, the kits may include one or moreampoules or syringes that contain AAV particles or polynucleotides in asuitable liquid or solution form.

EXAMPLES Rescue of Hearing in OTOF Knock-Out Mice Using Adeno-AssociatedVirus Gene Therapy Approach Introduction

Otoferlin is the key calcium sensor for neurotransmitter release in theear (see, e.g., Roux 2006). Otoferlin is mainly expressed in the innerhair cells of the cochlea and only few other cells of the centralnervous system (see, e.g., Yasunaga et al. 1999& 2000). It is a memberof the ferlin family of transmembrane proteins which share a common C2domain also found in synaptotagmin, PKC and PLC.

Mutations in the human OTOF gene, which encodes human Otoferlin, cause atype of nonsyndromic deafness called Deafness, Autosomal Recessive 9(DFNB9). OTOF knock-out mice have also been shown to have severe hearingloss despite normal inner hair cell development and auditory ribbonsynapse formation (see, e.g., Roux et al. (2006) Otoferlin, defective ina human deafness form, is essential for exocytosis at the auditoryribbon synapse. Cell. 127:277-289). However, Otof^(−/−) mice lose theauditory brain stem response across all sound frequencies due tocomplete abolishment of synaptic exocytosis and, as a consequence,abolishment of neurotransmitter release from synaptic vesicles.

DFNB9 manifests in humans as two phenotypes, as a nonsyndromic bilateralloss of hearing before the acquiring of language and less frequently asa temperature-sensitive nonsyndromic auditory neuropathy. It was firstdiscovered in an affected Lebanese family (Chaib et al. 1996) and hassince been found in many parts of the world (see, e.g., Adato et al.2000, Rodriguez-Ballesteros et al. 2003, Choi et al. 2009, Matsunaga etal. 2012).

Current treatment in humans with DFNB9 utilizes cochlear implants andhearing aids. In addition, for the temperature-sensitive form of DFNB9,prevention of fevers and other conditions that would cause the bodytemperature to rise are important. Applicants sought to useadeno-associated virus (AAV) as a means to restore expression of OTOF inthe knock-out mice as a proof-of-concept for using AAV to delivery OTOFas a treatment for DFNB9. The mouse OTOF cDNA is 5979 base pairs inlength whereas most AAVs cannot package more than approximately 4.8kilobases of genome. As a result, a dual vector system was used toseparately deliver the 5′ portion of the cDNA and the 3′ portion of thecDNA as separate AAV constructs such that the full-length cDNA could bereassembled in vivo once delivered.

Methods Dual AAV Vector Constructs

A mouse OTOF cDNA was split into two sections, a 5′ and 3′ section andinserted into two AAV ITR-containing plasmids. The sequence of each ofthe two cassettes in the plasmids is shown below and the maps of eachconstruct are shown in FIGS. 1 and 2. Annotated versions of thecassettes are shown in FIGS. 3A and 3B. Each cassette contains a regionof homology to promote homologous recombination between the 5′ and 3′ends of the cDNA in vivo (see Ghosh et al., 2011). Once recombined invivo, the full-length cDNA contains a splice donor/splice acceptor pairthat causes splicing out of the region of homology. The vectors werepackaged into AAV2 serotype particles using standard plasmidtransfection methods as previously described (see Zolotukhin et al.Production and purification of serotype 1, 2, and 5 recombinantadeno-associated viral vectors. Methods 28 (2002) 158-167). The viralparticles were purified by standard methods as previously described (seeZolotukhin et al. Production and purification of serotype 1, 2, and 5recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167).The viral particles carrying the 5′ portion of the OTOF cDNA are alsoreferred to herein as “AAV2-OTOF-NT.” The viral particles carrying the3′ portion of the OTOF cDNA are also referred to herein as“AAV2-OTOF-CT.”

pTR22-smCBA-otoferlinNT-APSD-APhead (SEQ ID NO: 1)AGGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCAGATCTGGCGCGCCCAATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCTAGCGGCCGCCACCATGGCCCTGATTGTTCACCTCAAGACTGTCTCAGAGCTCCGAGGCAAAGGTGACCGGATTGCCAAAGTCACTTTCCGAGGGCAGTCTTTCTACTCCCGGGTCCTGGAGAACTGCGAGGGTGTGGCTGACTTTGATGAGACGTTCCGGTGGCCAGTGGCCAGCAGCATCGACCGGAATGAAGTGTTGGAGATTCAGATTTTCAACTACAGCAAAGTCTTCAGCAACAAGCTGATAGGGACCTTCTGCATGGTGCTGCAGAAAGTGGTGGAGGAGAATCGGGTAGAGGTGACCGACACGCTGATGGATGACAGCAATGCTATCATCAAGACCAGCCTGAGCATGGAGGTCCGGTATCAGGCCACAGATGGCACTGTGGGCCCCTGGGATGATGGAGACTTCCTGGGAGATGAATCCCTCCAGGAGGAGAAGGACAGCCAGGAGACAGATGGGCTGCTACCTGGTTCCCGACCCAGCACCCGGATATCTGGCGAGAAGAGCTTTCGCAGCAAAGGCAGAGAGAAGACCAAGGGAGGCAGAGATGGCGAGCACAAAGCGGGAAGGAGTGTGTTCTCGGCCATGAAACTCGGCAAAACTCGGTCCCACAAAGAGGAGCCCCAAAGACAAGATGAGCCAGCAGTGCTGGAGATGGAGGACCTGGACCACCTAGCCATTCAGCTGGGGGATGGGCTGGATCCTGACTCCGTGTCTCTAGCCTCGGTCACCGCTCTCACCAGCAATGTCTCCAACAAACGGTCTAAGCCAGATATTAAGATGGAGCCCAGTGCTGGAAGGCCCATGGATTACCAGGTCAGCATCACAGTGATTGAGGCTCGGCAGCTGGTGGGCTTGAACATGGACCCTGTGGTGTGTGTGGAGGTGGGTGATGACAAGAAATACACGTCAATGAAGGAGTCCACAAACTGCCCTTACTACAACGAGTACTTTGTCTTCGACTTCCATGTCTCTCCTGATGTCATGTTTGACAAGATCATCAAGATCTCGGTTATCCATTCTAAGAACCTGCTTCGGAGCGGCACCCTGGTGGGTTCCTTCAAAATGGATGTGGGGACTGTGTATTCCCAGCCTGAACACCAGTTCCATCACAAATGGGCCATCCTGTCAGACCCCGATGACATCTCTGCTGGGTTGAAGGGTTATGTAAAGTGTGATGTCGCTGTGGTGGGCAAGGGAGACAACATCAAGACACCCCACAAGGCCAACGAGACGGATGAGGACGACATTGAAGGGAACTTGCTGCTCCCCGAGGGCGTGCCCCCCGAACGGCAGTGGGCACGGTTCTATGTGAAAATTTACCGAGCAGAGGGACTGCCCCGGATGAACACAAGCCTCATGGCCAACGTGAAGAAGGCGTTCATCGGTGAGAACAAGGACCTCGTCGACCCCTATGTGCAAGTCTTCTTTGCTGGACAAAAGGGCAAAACATCAGTGCAGAAGAGCAGCTATGAGCCGCTATGGAATGAGCAGGTCGTCTTCACAGACTTGTTCCCCCCACTCTGCAAACGCATGAAGGTGCAGATCCGGGACTCTGACAAGGTCAATGATGTGGCCATCGGCACCCACTTCATCGACCTGCGCAAGATTTCCAACGATGGAGACAAAGGCTTCCTGCCTACCCTCGGTCCAGCCTGGGTGAACATGTACGGCTCCACGCGCAACTACACACTGCTGGACGAGCACCAGGACTTGAATGAAGGCCTGGGGGAGGGTGTGTCCTTCCGGGCCCGCCTCATGTTGGGACTAGCTGTGGAGATCCTGGACACCTCCAACCCAGAGCTCACCAGCTCCACGGAGGTGCAGGTGGAGCAGGCCACGCCTGTCTCGGAGAGCTGCACAGGGAGAATGGAAGAATTTTTTCTATTTGGAGCCTTCTTGGAAGCCTCAATGATTGACCGGAAAAATGGGGACAAGCCAATTACCTTTGAGGTGACCATAGGAAACTACGGCAATGAAGTCGATGGTATGTCCCGGCCCCTGAGGCCTCGGCCCCGGAAAGAGCCTGGGGATGAAGAAGAGGTAGACCTGATTCAGAACTCCAGTGACGATGAAGGTGACGAAGCCGGGGACCTGGCCTCGGTGTCCTCCACCCCACCTATGCGGCCCCAGATCACGGACAGGAACTATTTCCACCTGCCCTACCTGGAGCGCAAGCCCTGCATCTATATCAAGAGCTGGTGGCCTGACCAGAGGCGGCGCCTCTACAATGCCAACATCATGGATCACATTGCTGACAAGCTGGAAGAAGGCCTGAATGATGTACAGGAGATGATCAAAACGGAGAAGTCCTACCCGGAGCGCCGCCTGCGGGGTGTGCTAGAGGAACTCAGCTGTGGCTGCCACCGCTTCCTCTCCCTCTCGGACAAGGACCAGGGCCGCTCGTCCCGCACCAGGCTGGATCGAGAGCGTCTTAAGTCCTGTATGAGGGAGTTGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGAGCTAGCCCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCGTCGACTGTTAATTAAGCATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGGCTG CpTR22-APhead-APSA-otoferlinCT (SEQ ID NO: 2)AGGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCAGATCTGGCGCGCCCAATTGGCTTCGAATTCTAGCGGCCGCCCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCCTTAAGCGACGCATGCTCGCGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGAGAGCATGGGACAGCAGGCCAAGAGCCTGAGGGCTCAGGTGAAGCGGCACACTGTTCGGGACAAGCTGAGGTCATGCCAGAACTTTCTGCAGAAGCTACGCTTCCTGGCGGATGAGCCCCAGCACAGCATTCCTGATGTGTTCATTTGGATGATGAGCAACAACAAACGTATCGCCTATGCCCGCGTGCCTTCCAAAGACCTGCTCTTCTCCATCGTGGAGGAGGAACTGGGCAAGGACTGCGCCAAAGTCAAGACCCTCTTCCTGAAGCTGCCAGGGAAGAGGGGCTTCGGCTCGGCAGGCTGGACAGTACAGGCCAAGCTGGAGCTCTACCTGTGGCTGGGCCTCAGCAAGCAGCGAAAGGACTTCCTGTGTGGTCTGCCCTGTGGCTTCGAGGAGGTCAAGGCAGCCCAAGGCCTGGGCCTGCATTCCTTTCCGCCCATCAGCCTAGTCTACACCAAGAAGCAAGCCTTCCAGCTCCGAGCACACATGTATCAGGCCCGAAGCCTCTTTGCTGCTGACAGCAGTGGGCTCTCTGATCCCTTTGCCCGTGTCTTCTTCATCAACCAGAGCCAATGCACTGAGGTTCTAAACGAGACACTGTGTCCCACCTGGGACCAGATGCTGGTATTTGACAACCTGGAGCTGTACGGTGAAGCTCACGAGTTACGAGATGATCCCCCCATCATTGTCATTGAAATCTACGACCAGGACAGCATGGGCAAAGCCGACTTCATGGGCCGGACCTTCGCCAAGCCCCTGGTGAAGATGGCAGATGAAGCATACTGCCCACCTCGCTTCCCGCCGCAGCTTGAGTACTACCAGATCTACCGAGGCAGTGCCACTGCCGGAGACCTACTGGCTGCCTTCGAGCTGCTGCAGATTGGGCCATCAGGGAAGGCTGACCTGCCACCCATCAATGGCCCAGTGGACATGGACAGAGGGCCCATCATGCCTGTGCCCGTGGGAATCCGGCCAGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTGAGGGACCTAAAGAGGGTGAACCTGGCCCAGGTGGACCGACCACGGGTGGACATCGAGTGTGCAGGAAAGGGGGTACAATCCTCCCTGATTCACAATTATAAGAAGAACCCCAACTTCAACACGCTGGTCAAGTGGTTTGAAGTGGACCTCCCGGAGAATGAGCTCCTGCACCCACCCTTGAACATCCGAGTGGTAGATTGCCGGGCCTTTGGACGATACACCCTGGTGGGTTCCCACGCAGTCAGCTCACTGAGGCGCTTCATCTACCGACCTCCAGACCGCTCAGCCCCCAACTGGAACACCACAGGGGAGGTTGTAGTAAGCATGGAGCCTGAGGAGCCAGTTAAGAAGCTGGAGACCATGGTGAAACTGGATGCGACTTCTGATGCTGTGGTCAAGGTGGATGTGGCTGAAGATGAGAAGGAAAGGAAGAAGAAGAAAAAGAAAGGCCCGTCAGAGGAGCCAGAGGAGGAAGAGCCCGATGAGAGCATGCTGGATTGGTGGTCCAAGTACTTCGCCTCCATCGACACAATGAAGGAGCAACTTCGACAACATGAGACCTCTGGAACTGACTTGGAAGAGAAGGAAGAGATGGAAAGCGCTGAGGGCCTGAAGGGACCAATGAAGAGCAAGGAGAAGTCCAGAGCTGCAAAGGAGGAGAAAAAGAAGAAAAACCAGAGCCCTGGCCCTGGCCAGGGATCGGAGGCTCCTGAGAAGAAGAAAGCCAAGATCGATGAGCTTAAGGTGTACCCCAAGGAGCTGGAATCGGAGTTTGACAGCTTTGAGGACTGGCTGCACACCTTCAACCTGTTGAGGGGCAAGACGGGAGATGATGAGGATGGCTCCACAGAGGAGGAGCGCATAGTAGGCCGATTCAAGGGCTCCCTCTGTGTGTACAAAGTGCCACTCCCAGAAGATGTATCTCGAGAAGCTGGCTATGATCCCACCTATGGAATGTTCCAGGGCATCCCAAGCAATGACCCCATCAATGTGCTGGTCCGAATCTATGTGGTCCGGGCCACAGACCTGCACCCGGCCGACATCAATGGCAAAGCTGACCCCTATATTGCCATCAAGTTAGGCAAGACCGACATCCGAGACAAGGAGAACTACATCTCCAAGCAGCTCAACCCTGTGTTTGGGAAGTCCTTTGACATTGAGGCCTCCTTCCCCATGGAGTCCATGTTGACAGTGGCCGTGTACGACTGGGATCTGGTGGGCACTGATGACCTCATCGGAGAAACCAAGATTGACCTGGAAAACCGCTTCTACAGCAAGCATCGCGCCACCTGCGGCATCGCACAGACCTATTCCATACATGGCTACAATATCTGGAGGGACCCCATGAAGCCCAGCCAGATCCTGACACGCCTCTGTAAAGAGGGCAAAGTGGACGGCCCCCACTTTGGTCCCCATGGGAGAGTGAGGGTTGCCAACCGTGTCTTCACGGGGCCTTCAGAAATAGAGGATGAGAATGGTCAGAGGAAGCCCACAGATGAGCACGTGGCACTGTCTGCTCTGAGACACTGGGAGGACATCCCCCGGGTGGGCTGCCGCCTTGTGCCGGAACACGTGGAGACCAGGCCGCTGCTCAACCCTGACAAGCCAGGCATTGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACACCTCTGGATATATCCCCCAGGAAACCCAAGAAGTACGAGCTGCGGGTCATCGTGTGGAACACAGACGAGGTGGTCCTGGAAGACGATGATTTCTTCACGGGAGAGAAGTCCAGTGACATTTTTGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAACAGGACACAGATGTCCACTATCACTCCCTCACGGGGGAGGGCAACTTCAACTGGAGATACCTCTTCCCCTTCGACTACCTAGCGGCCGAAGAGAAGATCGTTATGTCCAAAAAGGAGTCTATGTTCTCCTGGGATGAGACGGAGTACAAGATCCCTGCGCGGCTCACCCTGCAGATCTGGGACGCTGACCACTTCTCGGCTGACGACTTCCTGGGGGCTATCGAGCTGGACCTGAACCGGTTCCCGAGGGGCGCTAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTACCCCTGGTTTCCATCTTTAAACAGAAACGTGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAATGATGAGTTTGAGCTCACAGGCAAAGTGGAGGCGGAGCTACACCTACTCACGGCAGAGGAGGCAGAGAAGAACCCTGTGGGCCTGGCTCGCAATGAACCTGATCCCCTAGAAAAACCCAACCGGCCTGACACGGCATTCGTCTGGTTCCTGAACCCACTCAAATCTATCAAGTACCTCATCTGCACCCGGTACAAGTGGCTGATCATCAAGATCGTGCTGGCGCTGCTGGGGCTGCTCATGCTGGCCCTCTTCCTTTACAGCCTCCCAGGCTACATGGTCAAGAAGCTCCTAGGGGCCTGAGCGGCCGCGGTACCAAGGGCGAATTCTGCAGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGAGATCTGAGGACTAGTCCGTCGACTGTTAATTAAGCATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTC TTCGCTATTACGCCAGGCTGC

Transfection of HEK 293 Cells

HEK 293 cells were grown on poly-lysine-coated coverslips in growthmedium. 1 μl of each virus was used for each well as follows: Controlcells without virus, cells with AAV2-OTOF n-terminal portion(AAV2-OTOF-NT), cells with AAV2-OTOF c-terminal portion (AAV2-OTOF-CT)and cells with both viruses (AAV2-OTOF-NT and AAV2-OTOF-CT). The cellswere stained with anti-OTOF antibody and mounted on glass slides.

OTOF Knock-Out Mice

The OTOF knock-out mice used were generated in a previous study (seeRoux et al. (2006) Otoferlin, defective in a human deafness form, isessential for exocytosis at the auditory ribbon synapse. Cell.127:277-289). Briefly, two fragments containing the genomic sequences 5′and 3′ to exons 14 and 15 of Otof were amplified by PCR. The 5 kbBamHI-XhoI-BssHII and 6 kb BssHII-SfiI-BamHI-NaeI 129/SvPas fragmentswere inserted into pUC19 (New England BioLabs) previously modified byinserting a BamHI-XhoI-BssHII-SfiI-NaeI-HindIII polylinker. AloxP-hygro-loxP (the gene conferring resistance to hygromycin undercontrol of the phosphoglycerate kinase gene [Pgk-1] promoter) cassettewas inserted into the BssHII site. All constructs were sequenced, andthe sequences obtained were compared with the 129/SvPas genomic sequence282 CK35 ES cells resistant to hygromycin were screened for homologousrecombination and monoinsertion events by Southern blot analysis. Twoclones were injected into C57BL/6N blastocysts to create chimericanimals. Transmission of the mutant Otof allele was detected by PCR inagouti pups. Positive pups in the F1 progeny were crossed with Pgk-1-cremice in a mixed C57BL/6-129/SvPas background. F2 animals carrying anallele in which the hygromycin selection cassette was deleted(Otoftm1Ugds allele) were selected by PCR, using primers5′-CACTTGCTTTGTCT CATCTCC-3′ (SEQ ID NO: 12) and5′-GTCACTTCTTCTGGGTATTTC-3′ (SEQ ID NO: 13), generating a 507 base pairPCR product. The heterozygous animals were interbred to generateOtof^(−/−), Otof^(+/−), and Otof^(+/+) mice. The knockout mice weregenerated in C57BL/6-129/SvPas background as described above. Becausethis background strain is known to have some age-related hearing loss,the mice were backcrossed with FVB mice strain to the 10^(th) generationto obtain an FVB homogeneous genetic background with no knownage-related hearing loss.

Delivery of AAV to Mice

OTOF knock-out mice (newborn and older than P10) were injected with 1microliter of viral particles of each of the two AAV constructs using around window membrane (RWM) injection as previously described (see Akilet al. (2012) Restoration of Hearing in the VGLUT3 Knockout Mouse UsingVirally-Mediated Gene Therapy. Neuron. 75(2): 283-293 and Akil et al.(2015) Surgical Method for Virally Mediated Gene Delivery to the MouseInner Ear through the Round Window Membrane. J. Vis. Exp. (97), e52187).AAV2-OTOF-NT (6.32×10¹²vg/ml) and AAV2-OTOE-CT (4.5×10¹²vg/ml) weredelivered through the RWM to P1-3 mice. AAV2-OTOF-NT (1.43×10¹³vg/ml)and AAV2-OTOF-CT (3.12×10¹³vg/ml) were delivered through the RWM to P>12mice. ABR tests were conducted 7 days after injection. Expression ofOTOF protein in the mice was measured using anti-OTOF antibody to labelcells in cochlear whole mounts. Reverse-transcriptase (RT)-PCR was usedto screen for the presence of OTOF mRNA within the cochlear tissue ofthe mice. Wild-type mice were also injected using the same techniquewith AAV2-GFP to assess viral delivery to cochlea with AAV2 serotype.Cochlea were whole mounted and stained with anti-GFP antibody.

Auditory Brainstem Response (ABR) Testing

Hearing tests were performed as previously described (Akil et al. (2006)Progressive deafness and altered cochlear innervation in knockout micelacking prosaposin. J. Neurosci. 26:13076-13088 and Akil et al. (2016)Mouse Auditory Brainstem Response Testing. Bio Protoc. 6(6)) with theotoferlin knockout (OTOF KO) mice, rescued OTOF KO mice and wild-type(WT) littermates. Briefly, all auditory testing was performed in asound-proof chamber. Before acoustic testing, mice were anesthetized byintraperitoneal injection of a mixture of ketamine hydrochloride(Ketaset, 100 mg/ml) and xylazine hydrochloride (xyla-ject, 10 mg/ml)and boosted with one-fifth the original dose as required. Bodytemperature was maintained with a heating pad and monitored with arectal probe throughout recording.

The evoked acoustic brainstem response (ABR) thresholds weredifferentially recorded from the scalp of the mice. Responses wererecorded using subdermal needle electrodes at the vertex, below thepinna of the left ear (reference), and below the contralateral ear(ground). The sound stimuli used included clicks (5 ms duration, 31 Hz)and tone pips at 8, 16, and 32 kHz (10 ms duration, cos 2 shaping, 21Hz). Measurements were recorded using the TDT BioSig III system (TuckerDavis Technologies). For each stimulus, electroencephalographic (EEG)activity was recorded for 20 ms (at a sampling rate of 25 kHz) andfiltered (0.3-3 kHz). Waveforms from 512 stimuli were averaged for clickresponses. Waveforms from 1000 stimuli were examined to identifyfrequency-specific tone-burst stimuli (8, 16, and 32 kHz). ABR waveformswere recorded in 5 dB sound pressure level (SPL) intervals down from themaximum amplitude. The threshold was defined as the lowest stimuluslevel at which response peaks for waves I-V were clearly andrepetitively present upon visual inspection. These threshold judgmentswere confirmed by analysis of stored waveforms. The comparison of eachgroup of animals was performed using one way ANOVA with Bonferroni'spost hoc testing.

Results

Two different AAV plasmid constructs were generated to deliver the 5′half and the 3′ half of the mouse OTOF cDNA to the inner ear of OTOFknock-out mice (OTOF N-terminal virus and OTOF C-terminal virus). Thetwo constructs were packaged separately into AAV2 particles. The AAV2particles were then pooled together and used to treat HEK 293 cells orwere injected into the inner ear of OTOF knock-out mice.

It was shown that HEK 293 cells only expressed Otoferlin protein whentransfected with both viruses (FIG. 4). No expression of Otoferlinprotein was observed in untreated cells, or in cells transfected withonly the OTOF N-terminal (FIG. 4) or OTOF C-terminal virus.

Next, the ability of AAV2 to transduce the mouse cochlea was assessusing an AAV2-GFP reporter virus. It was shown that AAV2 transfects anumber of cell types including inner hair cells (IHC), outer hair cells(OHC), pillar cells (P) and other supporting cells (SC) in the organ ofCorti (FIG. 5). Thus, AAV2 can transduce mouse cochlea effectively.

Mice were then treated with the pooled OTOF N-terminal and C-terminalviruses and compared to various controls. Otoferlin protein was found tobe expressed upon treatment with both viruses (FIG. 6). The largestnumber of transfected inner hair cells (IHCs) were observed in the baseand fewer in the mid-turn and apex (FIG. 6). IHCs counts demonstratedthat ˜11% of IHCs were labeled overall, with significant differencesseen between the base (˜29%), mid-turn (˜8%), and apex (˜2%). (FIG. 6).RT-PCR was used to show that OTOF mRNA was in whole cochlear extract andwas the same size in both wild-type and OTOF knock-out mice treated withboth viruses (FIG. 6). In contrast, the untreated OTOF knock-out mousecochleae did not demonstrate OTOF mRNA expression. No products weredetected when RT-PCR was performed in the absence of reversetranscriptase.

Next, hearing tests were performed to determine whether the Otoferlinexpressed by the delivery of both the N- and C-terminal viruses wascapable of rescuing hearing function. ABR waveforms from the wild-typeand the OTOF knock-out mice treated with both viruses were similar,documenting hearing recovery in the rescued KO mice whereas untreatedOTOF knock-out mice controls and the OTOF knock-out mice transfectedwith just OTOF N-terminal virus show no hearing recovery (FIG. 7). AtP70, partial hearing recovery (improved ABR thresholds) was seen toclicks and at specific frequencies 8, 16 and 32 kHz in the OTOFknock-out mice treated with both viruses, while at 8 and 16 kHz the ABRthresholds appear to be slightly elevated, though still significantlybetter than untreated OTOF knock-out mice (FIG. 7). Remarkably, hearingwas maintained in the OTOF knock-out mice treated with both viruses (KONT+CT) for more than 4 months (FIG. 7), although ABR thresholds weresomewhat variable. Non-transfected KO controls and the KO transfectedwith OTOF NT remained deaf (FIG. 7).

Next, OTOF knock-out mice older than P12 treated with both viruses. Thedually transfected IHCs expressed OTOF, with homogenous transfectionrates seen in the base (not shown) and the apex (FIG. 8). IHCs countsdemonstrated that ˜41% of IHCs were labeled overall, with slightdifferences seen between the base (˜38%), mid-turn (˜42%), and apex(˜47%) (FIG. 8).

At P60 all OTOF knock-out mice treated with both viruses demonstratednormal ABR threshold to clicks stimulus while at specific frequencies 8,16 and 32 kHz the ABR thresholds appeared to be slightly elevated,though still significantly better than untreated OTOF knock-out mice(FIG. 9). A time course of hearing recovery following injection of bothviruses into OTOF knock-out mice at an age older than P12 showed thathearing was maintained in the treated mice for more than 30 weeks, andthe ABR thresholds were restored to the WT levels (FIG. 9).

These results demonstrate that a use of more than one AAV construct todeliver different parts of an OTOF cDNA can result in a functional cDNAin vivo. These results also demonstrate that hearing loss can be treatedby delivery of OTOF cDNA using an AAV delivery system.

Example 2: Human OTOF Dual Vector Constructs

Provided below are example dual vector sequences for expressing HumanOtoferlin protein isoforms 1 and 5. The cDNAs encoding both isoforms 1and 5 contain the same N-terminal sequence such that the same N-terminalvector can be used for expressing both isoforms. Vector maps andannotated sequences corresponding to the below sequences are shown inFIGS. 10-15.

pTR22-smCBA-otoferlinNT Hs var 1 + 5-APSD-APhead (SEQ ID NO: 14)AGGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCAGATCTGGCGCGCCCAATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCTAGCGGCCGCCACCATGGCCTTGCTCATCCACCTCAAGACAGTCTCGGAGCTGCGGGGCAGGGGCGACCGGATCGCCAAAGTGACTTTCCGAGGGCAATCCTTCTACTCTCGGGTCCTGGAGAACTGTGAGGATGTGGCTGACTTTGATGAGACATTTCGGTGGCCGGTGGCCAGCAGCATCGACAGAAATGAGATGCTGGAGATTCAGGTTTTCAACTACAGCAAAGTCTTCAGCAACAAGCTCATCGGGACCTTCCGCATGGTGCTGCAGAAGGTGGTAGAGGAGAGCCATGTGGAGGTGACTGACACGCTGATTGATGACAACAATGCTATCATCAAGACCAGCCTGTGCGTGGAGGTCCGGTATCAGGCCACTGACGGCACAGTGGGCTCCTGGGACGATGGGGACTTCCTGGGAGATGAGTCTCTTCAAGAGGAAGAGAAGGACAGCCAAGAGACGGATGGACTGCTCCCAGGCTCCCGGCCCAGCTCCCGGCCCCCAGGAGAGAAGAGCTTCCGGAGAGCCGGGAGGAGCGTGTTCTCCGCCATGAAGCTCGGCAAAAACCGGTCTCACAAGGAGGAGCCCCAAAGACCAGATGAACCGGCGGTGCTGGAGATGGAAGACCTTGACCATCTGGCCATTCGGCTAGGAGATGGACTGGATCCCGACTCGGTGTCTCTAGCCTCAGTCACAGCTCTCACCACTAATGTCTCCAACAAGCGATCTAAGCCAGACATTAAGATGGAGCCAAGTGCTGGGCGGCCCATGGATTACCAGGTCAGCATCACGGTGATCGAGGCCCGGCAGCTGGTGGGCTTGAACATGGACCCTGTGGTGTGCGTGGAGGTGGGTGACGACAAGAAGTACACATCCATGAAGGAGTCCACTAACTGCCCCTATTACAACGAGTACTTCGTCTTCGACTTCCATGTCTCTCCGGATGTCATGTTTGACAAGATCATCAAGATTTCGGTGATTCACTCCAAGAACCTGCTGCGCAGTGGCACCCTGGTGGGCTCCTTCAAAATGGACGTGGGAACCGTGTACTCGCAGCCAGAGCACCAGTTCCATCACAAGTGGGCCATCCTGTCTGACCCCGATGACATCTCCTCGGGGCTGAAGGGCTACGTGAAGTGTGACGTTGCCGTGGTGGGCAAAGGGGACAACATCAAGACGCCCCACAAGGCCAATGAGACCGACGAAGATGACATTGAGGGGAACTTGCTGCTCCCCGAGGGGGTGCCCCCCGAACGCCAGTGGGCCCGGTTCTATGTGAAAATTTACCGAGCAGAGGGGCTGCCCCGTATGAACACAAGCCTCATGGCCAATGTAAAGAAGGCTTTCATCGGTGAAAACAAGGACCTCGTGGACCCCTACGTGCAAGTCTTCTTTGCTGGCCAGAAGGGCAAGACTTCAGTGCAGAAGAGCAGCTATGAGCCCCTGTGGAATGAGCAGGTCGTCTTTACAGACCTCTTCCCCCCACTCTGCAAACGCATGAAGGTGCAGATCCGAGACTCGGACAAGGTCAACGACGTGGCCATCGGCACCCACTTCATTGACCTGCGCAAGATTTCTAATGACGGAGACAAAGGCTTCCTGCCCACACTGGGCCCAGCCTGGGTGAACATGTACGGCTCCACACGTAACTACACGCTGCTGGATGAGCATCAGGACCTGAACGAGGGCCTGGGGGAGGGTGTGTCCTTCCGGGCCCGGCTCCTGCTGGGCCTGGCTGTGGAGATCGTAGACACCTCCAACCCTGAGCTCACCAGCTCCACAGAGGTGCAGGTGGAGCAGGCCACGCCCATCTCGGAGAGCTGTGCAGGTAAAATGGAAGAATTCTTTCTCTTTGGAGCCTTCCTGGAGGCCTCAATGATCGACCGGAGAAACGGAGACAAGCCCATCACCTTTGAGGTCACCATAGGCAACTATGGGAACGAAGTTGATGGCCTGTCCCGGCCCCAGCGGCCTCGGCCCCGGAAGGAGCCGGGGGATGAGGAAGAAGTAGACCTGATTCAGAACGCAAGTGATGACGAGGCCGGTGATGCCGGGGACCTGGCCTCAGTCTCCTCCACTCCACCAATGCGGCCCCAGGTCACCGACAGGAACTACTTCCATCTGCCCTACCTGGAGCGAAAGCCCTGCATCTACATCAAGAGCTGGTGGCCGGACCAGCGCCGCCGCCTCTACAATGCCAACATCATGGACCACATTGCCGACAAGCTGGAAGAAGGCCTGAACGACATACAGGAGATGATCAAAACGGAGAAGTCCTACCCTGAGCGTCGCCTGCGGGGCGTCCTGGAGGAGCTGAGCTGTGGCTGCTGCCGCTTCCTCTCCCTCGCTGACAAGGACCAGGGCCACTCATCCCGCACCAGGCTTGACCGGGAGCGCCTCAAGTCCTGCATGAGGGAGCTGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGAGCTAGCCCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCGTCGACTGTTAATTAAGCATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCC AGGCTGCpTR22-APhead-APSA-otoferlinCT Hs var 1 (SEQ ID NO: 15)AGGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCAGATCTGGCGCGCCCAATTGGCTTCGAATTCTAGCGGCCGCCCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCCTTAAGCGACGCATGCTCGCGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGAAAACATGGGGCAGCAGGCCAGGATGCTGCGGGCCCAGGTGAAGCGGCACACGGTGCGGGACAAGCTGAGGCTGTGCCAGAACTTCCTGCAGAAGCTGCGCTTCCTGGCGGACGAGCCCCAGCACAGCATTCCCGACATCTTCATCTGGATGATGAGCAACAACAAGCGTGTCGCCTATGCCCGTGTGCCCTCCAAGGACCTGCTCTTCTCCATCGTGGAGGAGGAGACTGGCAAGGACTGCGCCAAGGTCAAGACGCTCTTCCTTAAGCTGCCAGGGAAGCGGGGCTTCGGCTCGGCAGGCTGGACAGTGCAGGCCAAGGTGGAGCTGTACCTGTGGCTGGGCCTCAGCAAACAGCGCAAGGAGTTCCTGTGCGGCCTGCCCTGTGGCTTCCAGGAGGTCAAGGCAGCCCAGGGCCTGGGCCTGCATGCCTTCCCACCCGTCAGCCTGGTCTACACCAAGAAGCAGGCGTTCCAGCTCCGAGCGCACATGTACCAGGCCCGCAGCCTCTTTGCCGCCGACAGCAGCGGACTCTCAGACCCCTTTGCCCGCGTCTTCTTCATCAATCAGAGTCAGTGCACAGAGGTGCTGAATGAGACCCTGTGTCCCACCTGGGACCAGATGCTGGTGTTCGACAACCTGGAGCTCTATGGTGAAGCTCATGAGCTGAGGGACGATCCGCCCATCATTGTCATTGAAATCTATGACCAGGATTCCATGGGCAAAGCTGACTTCATGGGCCGGACCTTCGCCAAACCCCTGGTGAAGATGGCAGACGAGGCGTACTGCCCACCCCGCTTCCCACCTCAGCTCGAGTACTACCAGATCTACCGTGGCAACGCCACAGCTGGAGACCTGCTGGCGGCCTTCGAGCTGCTGCAGATTGGACCAGCAGGGAAGGCTGACCTGCCCCCCATCAATGGCCCGGTGGACGTGGACCGAGGTCCCATCATGCCCGTGCCCATGGGCATCCGGCCCGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTACGGGACCTAAAGCGGGTGAACCTGGCCCAGGTGGACCGGCCACGGGTGGACATCGAGTGTGCAGGGAAGGGGGTGCAGTCGTCCCTGATCCACAATTATAAGAAGAACCCCAACTTCAACACCCTCGTCAAGTGGTTTGAAGTGGACCTCCCAGAGAACGAGCTGCTGCACCCGCCCTTGAACATCCGTGTGGTGGACTGCCGGGCCTTCGGTCGCTACACACTGGTGGGCTCCCATGCCGTCAGCTCCCTGCGACGCTTCATCTACCGGCCCCCAGACCGCTCGGCCCCCAGCTGGAACACCACGGTCAGGCTTCTCCGGCGCTGCCGTGTGCTGTGCAATGGGGGCTCCTCCTCTCACTCCACAGGGGAGGTTGTGGTGACTATGGAGCCAGAGGTACCCATCAAGAAACTGGAGACCATGGTGAAGCTGGACGCGACTTCTGAAGCTGTTGTCAAGGTGGATGTGGCTGAGGAGGAGAAGGAGAAGAAGAAGAAGAAGAAGGGCACTGCGGAGGAGCCAGAGGAGGAGGAGCCAGACGAGAGCATGCTGGACTGGTGGTCCAAGTACTTTGCCTCCATTGACACCATGAAGGAGCAACTTCGACAACAAGAGCCCTCTGGAATTGACTTGGAGGAGAAGGAGGAAGTGGACAATACCGAGGGCCTGAAGGGGTCAATGAAGGGCAAGGAGAAGGCAAGGGCTGCCAAAGAGGAGAAGAAGAAGAAAACTCAGAGCTCTGGCTCTGGCCAGGGGTCCGAGGCCCCCGAGAAGAAGAAACCCAAGATTGATGAGCTTAAGGTATACCCCAAAGAGCTGGAGTCCGAGTTTGATAACTTTGAGGACTGGCTGCACACTTTCAACTTGCTTCGGGGCAAGACCGGGGATGATGAGGATGGCTCCACCGAGGAGGAGCGCATTGTGGGACGCTTCAAGGGCTCCCTCTGCGTGTACAAAGTGCCACTCCCAGAGGACGTGTCCCGGGAAGCCGGCTACGACTCCACCTACGGCATGTTCCAGGGCATCCCGAGCAATGACCCCATCAATGTGCTGGTCCGAGTCTATGTGGTCCGGGCCACGGACCTGCACCCTGCTGACATCAACGGCAAAGCTGACCCCTACATCGCCATCCGGCTAGGCAAGACTGACATCCGCGACAAGGAGAACTACATCTCCAAGCAGCTCAACCCTGTCTTTGGGAAGTCCTTTGACATCGAGGCCTCCTTCCCCATGGAATCCATGCTGACGGTGGCTGTGTATGACTGGGACCTGGTGGGCACTGATGACCTCATTGGGGAAACCAAGATCGACCTGGAGAACCGCTTCTACAGCAAGCACCGCGCCACCTGCGGCATCGCCCAGACCTACTCCACACATGGCTACAATATCTGGCGGGACCCCATGAAGCCCAGCCAGATCCTGACCCGCCTCTGCAAAGACGGCAAAGTGGACGGCCCCCACTTTGGGCCCCCTGGGAGAGTGAAGGTGGCCAACCGCGTCTTCACTGGGCCCTCTGAGATTGAGGACGAGAACGGTCAGAGGAAGCCCACAGACGAGCATGTGGCGCTGTTGGCCCTGAGGCACTGGGAGGACATCCCCCGCGCAGGCTGCCGCCTGGTGCCAGAGCATGTGGAGACGAGGCCGCTGCTCAACCCCGACAAGCCGGGCATCGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACGCCTCTGGACATCTCACCTCGGAAGCCCAAGAAGTACGAGCTGCGGGTCATCATCTGGAACACAGATGAGGTGGTCTTGGAGGACGACGACTTCTTCACAGGGGAGAAGTCCAGTGACATCTTCGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAGCAGGACACAGACGTCCACTACCACTCCCTCACTGGCGAGGGCAACTTCAACTGGCGCTACCTGTTCCCCTTCGACTACCTGGCGGCGGAGGAGAAGATCGTCATCTCCAAGAAGGAGTCCATGTTCTCCTGGGACGAGACCGAGTACAAGATCCCCGCGCGGCTCACCCTGCAGATCTGGGATGCGGACCACTTCTCCGCTGACGACTTCCTGGGGGCCATCGAGCTGGACCTGAACCGGTTCCCGCGGGGCGCAAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTGCCCCTCGTGTCCATCTTCAAGCAAAAGCGCGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAACGATGAGTTTGAGCTCACGGGCAAGGTGGAGGCTGAGCTGCATTTACTGACAGCAGAGGAGGCAGAGAAGAACCCAGTGGGCCTGGCCCGCAATGAACCTGACCCCCTAGAGAAACCCAACCGGCCCGACACGAGCTTCATCTGGTTCCTGAACCCTCTCAAGTCGGCTCGCTACTTCTTGTGGCACACGTATCGCTGGCTGCTCCTCAAACTGTTGCTGCTCCTGCTGCTGCTCCTCCTCCTCGCCCTGTTCCTCTACTCTGTGCCTGGCTACCTGGTCAAGAAAATCCTCGGGGCCTGAGCGGCCGCGGTACCAAGGGCGAATTCTGCAGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGAGATCTGAGGACTAGTCCGTCGACTGTTAATTAAGCATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGGCTGC pTR22-APhead-APSA-otoferlinCT Hs var 5(SEQ ID NO: 16) AGGGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCAGATCTGGCGCGCCCAATTGGCTTCGAATTCTAGCGGCCGCCCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGCGCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTCCTTAAGCGACGCATGCTCGCGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGAAAACATGGGGCAGCAGGCCAGGATGCTGCGGGCCCAGGTGAAGCGGCACACGGTGCGGGACAAGCTGAGGCTGTGCCAGAACTTCCTGCAGAAGCTGCGCTTCCTGGCGGACGAGCCCCAGCACAGCATTCCCGACATCTTCATCTGGATGATGAGCAACAACAAGCGTGTCGCCTATGCCCGTGTGCCCTCCAAGGACCTGCTCTTCTCCATCGTGGAGGAGGAGACTGGCAAGGACTGCGCCAAGGTCAAGACGCTCTTCCTTAAGCTGCCAGGGAAGCGGGGCTTCGGCTCGGCAGGCTGGACAGTGCAGGCCAAGGTGGAGCTGTACCTGTGGCTGGGCCTCAGCAAACAGCGCAAGGAGTTCCTGTGCGGCCTGCCCTGTGGCTTCCAGGAGGTCAAGGCAGCCCAGGGCCTGGGCCTGCATGCCTTCCCACCCGTCAGCCTGGTCTACACCAAGAAGCAGGCGTTCCAGCTCCGAGCGCACATGTACCAGGCCCGCAGCCTCTTTGCCGCCGACAGCAGCGGACTCTCAGACCCCTTTGCCCGCGTCTTCTTCATCAATCAGAGTCAGTGCACAGAGGTGCTGAATGAGACCCTGTGTCCCACCTGGGACCAGATGCTGGTGTTCGACAACCTGGAGCTCTATGGTGAAGCTCATGAGCTGAGGGACGATCCGCCCATCATTGTCATTGAAATCTATGACCAGGATTCCATGGGCAAAGCTGACTTCATGGGCCGGACCTTCGCCAAACCCCTGGTGAAGATGGCAGACGAGGCGTACTGCCCACCCCGCTTCCCACCTCAGCTCGAGTACTACCAGATCTACCGTGGCAACGCCACAGCTGGAGACCTGCTGGCGGCCTTCGAGCTGCTGCAGATTGGACCAGCAGGGAAGGCTGACCTGCCCCCCATCAATGGCCCGGTGGACGTGGACCGAGGTCCCATCATGCCCGTGCCCATGGGCATCCGGCCCGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTACGGGACCTAAAGCGGGTGAACCTGGCCCAGGTGGACCGGCCACGGGTGGACATCGAGTGTGCAGGGAAGGGGGTGCAGTCGTCCCTGATCCACAATTATAAGAAGAACCCCAACTTCAACACCCTCGTCAAGTGGTTTGAAGTGGACCTCCCAGAGAACGAGCTGCTGCACCCGCCCTTGAACATCCGTGTGGTGGACTGCCGGGCCTTCGGTCGCTACACACTGGTGGGCTCCCATGCCGTCAGCTCCCTGCGACGCTTCATCTACCGGCCCCCAGACCGCTCGGCCCCCAGCTGGAACACCACGGTCAGGCTTCTCCGGCGCTGCCGTGTGCTGTGCAATGGGGGCTCCTCCTCTCACTCCACAGGGGAGGTTGTGGTGACTATGGAGCCAGAGGTACCCATCAAGAAACTGGAGACCATGGTGAAGCTGGACGCGACTTCTGAAGCTGTTGTCAAGGTGGATGTGGCTGAGGAGGAGAAGGAGAAGAAGAAGAAGAAGAAGGGCACTGCGGAGGAGCCAGAGGAGGAGGAGCCAGACGAGAGCATGCTGGACTGGTGGTCCAAGTACTTTGCCTCCATTGACACCATGAAGGAGCAACTTCGACAACAAGAGCCCTCTGGAATTGACTTGGAGGAGAAGGAGGAAGTGGACAATACCGAGGGCCTGAAGGGGTCAATGAAGGGCAAGGAGAAGGCAAGGGCTGCCAAAGAGGAGAAGAAGAAGAAAACTCAGAGCTCTGGCTCTGGCCAGGGGTCCGAGGCCCCCGAGAAGAAGAAACCCAAGATTGATGAGCTTAAGGTATACCCCAAAGAGCTGGAGTCCGAGTTTGATAACTTTGAGGACTGGCTGCACACTTTCAACTTGCTTCGGGGCAAGACCGGGGATGATGAGGATGGCTCCACCGAGGAGGAGCGCATTGTGGGACGCTTCAAGGGCTCCCTCTGCGTGTACAAAGTGCCACTCCCAGAGGACGTGTCCCGGGAAGCCGGCTACGACTCCACCTACGGCATGTTCCAGGGCATCCCGAGCAATGACCCCATCAATGTGCTGGTCCGAGTCTATGTGGTCCGGGCCACGGACCTGCACCCTGCTGACATCAACGGCAAAGCTGACCCCTACATCGCCATCCGGCTAGGCAAGACTGACATCCGCGACAAGGAGAACTACATCTCCAAGCAGCTCAACCCTGTCTTTGGGAAGTCCTTTGACATCGAGGCCTCCTTCCCCATGGAATCCATGCTGACGGTGGCTGTGTATGACTGGGACCTGGTGGGCACTGATGACCTCATTGGGGAAACCAAGATCGACCTGGAGAACCGCTTCTACAGCAAGCACCGCGCCACCTGCGGCATCGCCCAGACCTACTCCACACATGGCTACAATATCTGGCGGGACCCCATGAAGCCCAGCCAGATCCTGACCCGCCTCTGCAAAGACGGCAAAGTGGACGGCCCCCACTTTGGGCCCCCTGGGAGAGTGAAGGTGGCCAACCGCGTCTTCACTGGGCCCTCTGAGATTGAGGACGAGAACGGTCAGAGGAAGCCCACAGACGAGCATGTGGCGCTGTTGGCCCTGAGGCACTGGGAGGACATCCCCCGCGCAGGCTGCCGCCTGGTGCCAGAGCATGTGGAGACGAGGCCGCTGCTCAACCCCGACAAGCCGGGCATCGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACGCCTCTGGACATCTCACCTCGGAAGCCCAAGAAGTACGAGCTGCGGGTCATCATCTGGAACACAGATGAGGTGGTCTTGGAGGACGACGACTTCTTCACAGGGGAGAAGTCCAGTGACATCTTCGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAGCAGGACACAGACGTCCACTACCACTCCCTCACTGGCGAGGGCAACTTCAACTGGCGCTACCTGTTCCCCTTCGACTACCTGGCGGCGGAGGAGAAGATCGTCATCTCCAAGAAGGAGTCCATGTTCTCCTGGGACGAGACCGAGTACAAGATCCCCGCGCGGCTCACCCTGCAGATCTGGGATGCGGACCACTTCTCCGCTGACGACTTCCTGGGGGCCATCGAGCTGGACCTGAACCGGTTCCCGCGGGGCGCAAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTGCCCCTCGTGTCCATCTTCAAGCAAAAGCGCGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAACGATGAGTTTGAGCTCACGGGCAAGGTGGAGGCTGAGCTGCATTTACTGACAGCAGAGGAGGCAGAGAAGAACCCAGTGGGCCTGGCCCGCAATGAACCTGACCCCCTAGAGAAACCCAACCGGCCCGACACGGCCTTCGTCTGGTTCCTCAACCCTCTCAAGTCCATCAAGTACCTCATCTGCACCCGGTACAAGTGGCTCATCATCAAGATCGTGCTGGCGCTGTTGGGGCTGCTCATGTTGGGGCTCTTCCTCTACAGCCTCCCTGGCTACATGGTCAAAAAGCTCCTTGGGGCATGAGCGGCCGCGGTACCAAGGGCGAATTCTGCAGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGAGATCTGAGGACTAGTCCGTCGACTGTTAATTAAGCATGCTGGGGAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCCCCCCCTGCAGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGGCTGC

REFERENCES

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A.,    Shahzad, M., Husnain, T., Riazuddin, S., Griffith, A. J.,    Friedman, T. B. Identities and frequencies of mutations of the    otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan. Clin.    Genet. 2009 75: 237-243.-   5) Dong B, Nakai H, Xiao W. Characterization of genome integrity for    oversized recombinant AAV vector. Mol Ther. 2010 January;    18(1):87-92.-   6) Ghosh A, Yue Y, Duan D. Efficient transgene reconstitution with    hybrid dual AAV vectors carrying the minimized bridging sequences.    Hum Gene Ther. 2011 January; 22(1):77-83.-   7) Hirsch M L, Agbandje-McKenna M, Samulski R J. Little vector, big    gene transduction: fragmented genome reassembly of adeno-associated    virus. Mol Ther. 2010 January; 18(1):6-8.-   8) Lai Y, Yue Y, Duan D. Evidence for the failure of    adeno-associated virus serotype 5 to package a viral genome > or    =8.2 kb. 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OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of increasing expression of Otoferlin ina cell, the method comprising: contacting the cell with a first AAVparticle comprising a first polynucleotide; and contacting the cell witha second AAV particle comprising a second polynucleotide, wherein (i)the first polynucleotide comprises inverted terminal repeat sequencesflanking an expression cassette containing, from 5′ to 3′: (a) apromoter, (b) a partial coding sequence that encodes an N-terminalportion of an Otoferlin polypeptide, (c) a splice donor site, and (d) afirst region of homology containing a sequence that is homologous to asequence in the second polynucleotide, and (ii) the secondpolynucleotide comprises inverted terminal repeat sequences flanking anexpression cassette containing, from 5′ to 3′: (a) a second region ofhomology containing a sequence that is homologous to a sequence in thefirst polynucleotide, (b) a splice acceptor site, (c) a partial codingsequence that encodes a C-terminal portion of the Otoferlin polypeptide,and (d) a polyadenylation (pA) signal sequence.
 2. The method of claim1, wherein the region of homology in the first and secondpolynucleotides is between 50 and 500 nucleotides.
 3. The method ofclaim 2, wherein the region of homology in the first and secondpolynucleotides is between 50 and 300 nucleotides.
 4. The method ofclaim 3, wherein the region of homology comprises the nucleotidesequence of SEQ ID NO:
 3. 5. The method of any one of claims 1 to 4,wherein the promoter is a chimeric CMV β actin (smcBA) promoter.
 6. Themethod of claim 5, wherein the promoter comprises the sequence of SEQ IDNO:
 4. 7. The method of any one of claims 1 to 6, wherein the Otoferlinpolypeptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ IDNO:
 6. 8. The method of any one of claims 1 to 7, wherein the splicedonor site comprises the sequence of SEQ ID NO:
 7. 9. The method of anyone of claims 1 to 8, wherein the splice acceptor site comprises thesequence of SEQ ID NO:
 8. 10. The method of any one of claims 1 to 9,wherein the inverted terminal repeat sequences are AAV2 invertedterminal repeat sequences.
 11. The method of any one of claims 1 to 10,wherein the first and second AAV particle are AAV2 serotype particles.12. The method of any one of claims 1 to 11, wherein the cell is exvivo.
 13. The method of any one of claims 1 to 11, wherein the cell isin vivo.
 14. The method of claim 13, wherein the cell is in a mammaliansubject.
 15. The method of claim 14, wherein the subject has Deafness,Autosomal Recessive 9 (DFNB9).
 16. A composition comprising: a first AAVparticle comprising a first polynucleotide; and a second AAV particlecomprising a second polynucleotide, wherein (i) the first polynucleotidecomprises inverted terminal repeat sequences flanking an expressioncassette containing, from 5′ to 3′: (a) a promoter, (b) a partial codingsequence that encodes an N-terminal portion of an Otoferlin polypeptide,(c) a splice donor site, and (d) a first region of homology containing asequence that is homologous to a sequence in the second polynucleotide,and (ii) the second polynucleotide comprises inverted terminal repeatsequences flanking an expression cassette containing, from 5′ to 3′: (a)a second region of homology containing a sequence that is homologous toa sequence in the first polynucleotide, (b) a splice acceptor site, (c)a partial coding sequence that encodes a C-terminal portion of theOtoferlin polypeptide, and (d) a polyadenylation (pA) signal sequence.17. The composition of claim 16, wherein the region of homology in thefirst and second polynucleotides is between 50 and 500 nucleotides. 18.The composition of claim 17, wherein the region of homology in the firstand second polynucleotides is between 50 and 300 nucleotides.
 19. Thecomposition of claim 18, wherein the region of homology comprises thenucleotide sequence of SEQ ID NO:
 3. 20. The composition of any one ofclaims 16 to 19, wherein the promoter is a chimeric CMV β actin (smcBA)promoter.
 21. The composition of claim 20, wherein the promotercomprises the sequence of SEQ ID NO:
 4. 22. The composition of any oneof claims 16 to 21, wherein the Otoferlin polypeptide comprises theamino acid sequence of SEQ ID NO: 5 or SEQ ID NO:
 6. 23. The compositionof any one of claims 16 to 22, wherein the splice donor site comprisesthe sequence of SEQ ID NO:
 7. 24. The composition of any one of claims16 to 23, wherein the splice acceptor site comprises the sequence of SEQID NO:
 8. 25. The composition of any one of claims 16 to 24, wherein theinverted terminal repeat sequences are AAV2 inverted terminal repeatsequences.
 26. The composition of any one of claims 16 to 25, whereinthe first and second AAV particle are AAV2 serotype particles.
 27. Thecomposition of any one of claims 16 to 26, further comprising apharmaceutically acceptable carrier.
 28. A kit comprising thecomposition of any one of claims 16 to 27.